Recombinant Viral Vectors And Methods For Inducing A Heterosubtypic Immune Response To Influenza A Viruses

Abstract

The present invention relates to recombinant viral vectors and methods of using the recombinant viral vectors to induce an immune response to influenza A viruses. The invention provides recombinant viral vectors based, for example, on the non-replicating modified vaccinia virus Ankara. When administered according to methods of the invention, the recombinant viral vectors are designed to be cross-protective and induce heterosubtypic immunity to influenza A viruses.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to recombinant viral vectors and methods of using the recombinant viral vectors to induce an immune response to influenza A viruses. The invention provides recombinant viral vectors based, for example, on the non-replicating modified vaccinia virus Ankara. When administered according to methods of the invention, the recombinant viral vectors are designed to be cross-protective and induce heterosubtypic immunity to influenza A viruses.

BACKGROUND OF THE INVENTION

[0002] Human influenza or "the flu" is a respiratory disease that is caused by influenza A and B viruses. Epidemics of influenza cause significant illness and death worldwide each year, and vaccination is the most straightforward strategy to prevent infection and disease. Traditional influenza vaccines expose the recipient to influenza virus proteins causing the recipient to mount an immune response to the proteins. Proteins (or polypeptides) used in vaccines are commonly called "antigens." The commonly used seasonal influenza vaccines are based on the major antigen of the viruses, the hemagglutinin (HA). There are numerous influenza A subtypes having different HA antigens. Influenza A subtypes are divided and classified based on the HA and neuraminidase (NA) proteins that are expressed by the viruses. The influenza A subtype nomenclature is based on the HA subtype (of the sixteen different HA genes known in the art) and the NA subtype (of the nine different NA genes known in the art). Exemplary subtypes, include, but are not limited to, H5N1, H1N1 and H3N2. There are also variants of the influenza A subtypes which are referred to as "strains." For example, the virus A/VietNam/1203/2004 is an influenza A virus, subtype H5N1, with a strain name A/VietNam/1203/2004.

[0003] Protection from the seasonal vaccines based on the HA is strain-specific and new strains emerge constantly, so the classical influenza vaccines have to be re-formulated each year in an attempt to match the currently circulating strains. See, Lambert and Fauci 2010. It is therefore highly desirable for next generation vaccines to be cross-protective and induce heterosubtypic immunity, i.e., vaccines against one subtype that protect or partially protect against challenge infection with influenza A of different subtypes.

[0004] The current `universal vaccines` (i.e., vaccines designed to elicit heterosubtypic immunity) that are under development are mainly based on the more conserved internal influenza virus genes including the influenza matrix proteins (M1 and M2) (Schotsaert et al. 2009), the nucleoprotein (NP) and conserved parts of the HA (Bommakanti et al. 2010; Steel et al. 2010). The polymerase proteins PA, PB1 and PB2 also induce substantial T cell responses and may be also relevant targets (Assarsson et al. 2008; Greenbaum et al. 2009; Lee et al. 2008).

[0005] Next generation influenza vaccines currently under development include recombinant proteins, synthetic peptides, virus-like particles (VLPs), DNA-based vaccines and viral vector vaccines (Lambert and Fauci, supra). The advantage of using live viral vectors is their known property to induce high levels of cellular immunity, in particular CD8 T cells. Among the most promising viral vectors are vaccinia virus-based live vaccines (Rimmelzwaan and Sutter 2009) and adenovirus-based vectors (Hoelscher et al. 2006; Hoelscher et al. 2007; Price et al. 2010; Zhou et al. 2010). Single-dose mucosal immunization using an adenovirus construct expressing NP and M2, for instance, provided protection from virulent H5N1, H3N2 and H1N1 viruses (Price et al, supra). In a further study (Price et al. 2009), DNA vaccination with nucleoprotein (NP) and matrix 2 (M2) plasmids followed by boosting with antigen-matched recombinant adenovirus (rAd) provided robust protection against virulent H1N1 and H5N1 challenges in mice and ferrets.

[0006] Recombinant vaccines based on modified vaccinia virus Ankara (MVA) have been used in many non-clinical and clinical studies. MVA has proven to be exceptionally safe. No significant side effects have been obtained when MVA was administered to more than 120,000 human patients in the context of the smallpox eradication. Due to a block in virion morphogenesis the highly attenuated vaccinia virus strain fails to productively replicate in human and most other mammalian cells. Nevertheless, the ability to express viral and foreign genes in the early and late stage is retained. These characteristics make MVA a promising live vaccine vector that induces humoral and cellular immune responses and that exhibits a high safety profile.

[0007] U.S. Pat. Nos. 6,998,252; 7,015,024; 7,045,136 and 7,045,313 relate to recombinant poxviruses, such as vaccinia.

[0008] MVA-based vaccines have been used in clinical studies, for instance, against HIV, tuberculosis, malaria and cancer. In all of these studies, at least two doses were used. The human dose of an MVA-based vaccine was 5.times.10.sup.7 to 5.times.10.sup.8 PFU as applied in clinical trials (Brookes et al. 2008; Cebere et al. 2006; Tykodi and Thompson 2008;).

[0009] MVA has been used recently as a vector in pandemic H5N1 (Kreijtz et al. 2008; Kreijtz et al. PLoS One 2009; Kreijtz et al. Vaccine 2009; Kreijtz et al. J. Infect. Dis. 2009; Kreijtz et al. 2007; Mayrhofer et al., 2009; Poon et al. 2009) and H1N1 (Hessel et al. 2010; Kreijtz et al., J. Infect. Dis. 2009) influenza research. An MVA-based vaccine expressing NP and M1 is currently being tested in an ongoing clinical trial (Berthoud et al. 2011).

[0010] Thus, there remains a need in the art for a more broadly protective influenza vaccine.

DETAILED DESCRIPTION

[0011] The present invention provides recombinant viruses (also referred to as recombinant viral vectors herein) useful for generating a heterosubtypic immune response to influenza A viruses. The recombinant viruses are recombinant vaccinia viruses, such as recombinant MVA or other non-replicating or replicating vaccinia virus known in the art. Non-replicating vaccinia viruses include, but are not limited to, defective vaccinia Lister (dVV), MVA-575 (ECACC V00120707), MVA-BN (ECACC V00083008), MVA-F6 and MVA-M4 (Antoine et al. 1998). In some embodiments, the recombinant viruses encode a fusion protein (h1HA/M2e) comprising an influenza A hemagglutinin deletion mutant "headless HA" (h1HA) with at least one influenza A M2 external domain (M2e) insert; an h1HA/M2e fusion protein and an influenza A nucleoprotein (NP); or an h1HA and NP. The recombinant viruses of the invention may further encode an influenza A matrix protein 1 (M1) and/or an influenza A polymerase PB1. When administered according to methods of the invention, the recombinant viruses are cross-protective and induce heterosubtypic humoral and cellular immune responses (including CD8 and CD4 T cell responses). The recombinant viruses are therefore contemplated to be useful as universal influenza A vaccines in humans.

[0012] In some embodiments, the h1HA amino acid sequence encoded by an open reading frame in recombinant viruses of the invention may be, for example, the h1HA amino acid sequence set out in SEQ ID NO: 15 (based on A/VietNam/1203/2004 H5N1 HA NCBI Genbank AAW80717 which is SEQ ID NO: 3). The h1HA of SEQ ID NO: 15 comprises a signal sequence, the HA1 residues 17-58 of SEQ ID NO: 3, a linker peptide of four glycines, the HA1 residues 290-343 of SEQ ID NO: 3 and the HA2 stalk region residues 344-568 of SEQ ID NO: 3.

[0013] In some embodiments, the h1HA/M2e fusion protein amino acid sequence encoded by an open reading frame in recombinant viruses of the invention may be, for example, the h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO: 2. The fusion protein of SEQ ID NO: 2 comprises a signal sequence, the HA1 residues 17-58 of SEQ ID NO: 3, a linker peptide of three glycines (SEQ ID NO: 4), the M2e of H5N1 (SEQ ID NO: 5 based on A/VietNam/1203/2004 H5N1 NCBI Genbank ABP35634), a six-amino acid linker GSAGSA (SEQ ID NO: 9), the M2e of H1N1 (equivalent to H2N2 and H3N2) (SEQ ID NO: 6 based on A/New York/3315/2009 H1N1 NCBI Genbank ACZ05592), a six-amino acid linker GSAGSA (SEQ ID NO: 9), the M2e of H9N2 (SEQ ID NO: 7 based on A/chicken/Korea/SH0913/2009 H9N2 NCBI Genbank ADQ43641), a six-amino acid linker GSAGSA (SEQ ID NO: 9), the M2e of H7N2 (SEQ ID NO: 8 based on A/New York/107/2003 H7N2 NCBI Genbank ACC55276), a linker peptide of three glycines (SEQ ID NO: 4), the HA1 residues 290-343 of SEQ ID NO: 3 and the HA2 region residues 344-568 of SEQ ID NO: 3.

[0014] In some embodiments, the h1HA/M2e fusion protein may comprise one, two, three or four of the M2e polypeptides of SEQ ID NOs: 5, 6, 7 and 8. The h1HA/M2e fusion protein may comprise an influenza A M2e polypeptide other than an M2e polypeptide of SEQ ID NOs: 5, 6, 7, and 8.

[0015] In some embodiments, the NP amino acid sequence encoded by an open reading frame in recombinant viruses of the invention may be, for example, the NP amino acid sequence set out in SEQ ID NO: 13 (based on A/VietNam/1203/2004 H5N1 NP NCBI Genbank AAW80720). In some embodiments, the M1 amino acid sequence encoded by an open reading frame in recombinant viruses of the invention may be, for example, the M1 amino acid sequence set out in SEQ ID NO: 11 (based on A/VietNam/1203/2004 H5N1 M1 Genbank AAW80726). In some embodiments, the PB1 amino acid sequence encoded by an open reading frame in recombinant viruses of the invention may be, for example, the PB1 amino acid sequence set out in SEQ ID NO: 17 (based on A/VietNam/1203/2004 H5N1 PB1 Genbank AAW80711).

[0016] The invention contemplates that polypeptides encoded by an open reading frame in a recombinant virus may vary in sequence from SEQ ID NO: 2, 5, 6, 7, 8, 11, 13, 15 and/or 17 if the polypeptides retain the ability to induce a protective immune response when the recombinant virus is administered to an individual. In these embodiments, the polypeptide may be about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95%, about 97%, about 98% or about 99% identical to SEQ ID NO: 2, 5, 6, 7, 8, 11, 13, 15 and/or 17.

[0017] In other embodiments, h1HA/M2e fusion proteins, h1HA polypeptides and NP polypeptides encoded by recombinant viruses of the invention may be based on the same or different influenza A subtypes including, but not limited to, any combination of H1 to H16 and N1 to N9 (including H1N1, H2N1, H3N1, H4N1, H5N1, H6N1, H7N1, H8N1, H9N1, H10N1, H11N1, H12N1, H13N1, H14N1, H15N1, H16N1; H1N2, H2N2, H3N2, H4N2, H5N2, H6N2, H7N2, H8N2, H9N2, H10N2, H11N2, H12N2, H13N2, H14N2, H15N2, H16N2; H1N3, H2N3, H3N3, H4N3, H5N3, H6N3, H7N3, H8N3, H9N3, H10N3, H11N3, H12N3, H13N3, H14N3, H15N3, H16N3; H1N4, H2N4, H3N4, H4N4, H5N4, H6N4, H7N4, H8N4, H9N4, H10N4, H11N4, H12N4, H13N4, H14N4, H15N4, H16N4; H1N5, H2N5, H3N5, H4N5, H5N5, H6N5, H7N5, H8N5, H9N5, H10N5, H11N5, H12N5, H13N5, H14N5, H15N5, H16N5; H1N6, H2N6, H3N6, H4N6, H5N6, H6N6, H7N6, H8N6, H9N6, H10N6, H11N6, H12N6, H13N6, H14N6, H15N6, H16N6; H1N7, H2N7, H3N7, H4N7, H5N7, H6N7, H7N7, H8N7, H9N7, H10N7, H11N7, H12N7, H13N7, H14N7, H15N7, H16N7; H1N8, H2N8, H3N8, H4N8, H5N8, H6N8, H7N8, H8N8, H9N8, H10N8, H11N8, H12N8, H13N8, H14N8, H15N8, H16N8; H1N9, H2N9, H3N9, H4N9, H5N9, H6N9, H7N9, H8N9, H9N9, H10N9, H11N9, H12N9, H13N9, H14N9, H15N9, and H16N9). In some embodiments the influenza A subtype is a pandemic influenza A. Exemplary pandemic influenza subtypes include, but are not limited to, H1N1, H2N2, H3N2 and H5N1.

[0018] A list of identified Influenza A strains, including influenza A H1N1 strains, is available from the World Health Organization (WHO) and United States Centers for Disease Control (CDC) databases of Influenza A subtypes. The National Center for Biotechnology Information (NCBI) database maintained by the United States National Library of Medicine also maintains an updated database describing the length and sequence of HA, M2, NP, M1 and PB1 genes of viruses of influenza A species. Strains listed by these organizations and strains described in other commercial and academic databases, or in literature publications and known in the art, are contemplated for use in the invention. It is also contemplated that additional influenza A strains hereafter identified and isolated are also useful in the invention as sources of influenza A protein sequences. Accordingly, any strain specifically exemplified in the specification and those known or after discovered in the art are amenable to the recombinant vaccinia virus, pharmaceutical compositions, and methods of the invention. Exemplary strains include, but are not limited to, the strains in Table 1 below. The table also lists exemplary genes and associated database accession numbers of those strains.

TABLE-US-00001 TABLE 1 Inserted Virus Influenza NCBI gene NCBI amino acid Subtype gene Virus Strain acc no. acc no. H5N1 HA A/Viet Nam/1203/2004 AY818135 AAW80717 H5N1 NP A/Viet Nam/1203/2004 AY818138 AAW80720 H5N1 M1 A/Viet Nam/1203/2004 AY818144 AAW80726 H5N1 PB1 A/Viet Nam/1203/2004 AY818129 AAW80711 H5N1 M2 A/Viet Nam/1203/2004 EF541453 ABP35634 H1N1 sw M2 A/California/07/09 FJ969537 ACP44185 H1N1 M2 A/New York/3315/2009 CY050765 ACZ05592 H2N2 M2 A/Korea/426/68 NC_007377 YP_308853 H3N2 M2 A/New York/392/2004 NC_007367 YP_308840 H9N2 M2 A/chicken/Korea/SH0913/2009 HQ221654 ADQ43641 H7N2 M2 A/New York/107/2003 EU587373 ACC55276 H7N3 M2 A/chicken/Pakistan/34668/1995 CY035834 ACJ03948

[0019] In recombinant viruses of the invention, open reading frames encoding h1HA/M2e, h1HA, NP, M1 and/or PB1 may be codon-optimized for expression in human cells. In these embodiments, one or more (or all) of the naturally occurring codons in an open reading frame have been replaced in the codon-optimized open reading frame with codons frequently used in genes in human cells (sometimes referred to as preferred codons). Codons may be optimized to avoid repeat sequences to stabilize an open reading frame in the rMVA and/or to avoid unwanted transcription stop signals. Codon-optimization, in general, has been used in the field of recombinant gene expression to enhance expression of polypeptides in cells.

[0020] Gene cassettes encoding h1HA/M2e, h1HA, NP, M1 and PB1 in recombinant viruses of the invention include an open reading frame under the control of (i.e., operatively linked to) a promoter that functions (i.e., directs transcription of the open reading frame) in the recombinant vaccinia viruses. In exemplary embodiments, expression from gene cassettes is under the control of the strong early/late vaccinia virus mH5 promoter (SEQ ID NO: 18) or the synthetic early/late selP promoter (SEQ ID NO: 19) (Chakrabarti et al. 1997). In the gene cassettes of the invention the open reading frame is also operatively linked to a transcription stop signal such as a vaccinia virus early transcription stop signal.

[0021] In one aspect, the invention provides recombinant vaccinia virus comprising a gene cassette encoding an influenza A h1HA/M2e fusion protein. In some embodiments, the recombinant vaccinia virus is a recombinant MVA comprising a gene cassette expressing the h1HA/M2e fusion protein set out in SEQ ID NO: 2. In some embodiments, the recombinant vaccinia virus further comprises a gene cassette expressing the M1 protein (for example, the M1 set out in SEQ ID NO: 11) and/or a gene cassette expressing the PB1 protein (for example, the PB1 protein set out in SEQ ID NO: 17).

[0022] In another aspect, the invention provides recombinant vaccinia virus comprising a first gene cassette encoding an influenza A h1HA/M2e fusion protein, and a second gene cassette encoding an influenza NP. In some embodiments, the recombinant vaccinia virus is a recombinant MVA comprising a first gene cassette expressing the h1HA/M2e fusion protein set out in SEQ ID NO: 2 and a second gene cassette expressing the NP set out in SEQ ID NO: 13. In some embodiments, the recombinant vaccinia virus further comprises a gene cassette expressing the M1 protein (for example, the M1 set out in SEQ ID NO: 11) and/or a gene cassette expressing the PB1 protein (for example, the PB1 protein set out in SEQ ID NO: 17).

[0023] In yet another aspect, the invention provides recombinant vaccinia virus comprising a first gene cassette encoding an influenza A h1HA and a second gene cassette encoding an influenza NP. In some embodiments, the recombinant vaccinia virus is a recombinant MVA comprising a first gene cassette expressing the h1HA set out in SEQ ID NO: 15 and a second gene cassette expressing the NP set out in SEQ ID NO: 13. In some embodiments, the recombinant vaccinia virus further comprises a gene cassette expressing the M1 protein (for example, the M1 set out in SEQ ID NO: 11) and/or a gene cassette expressing the PB1 protein (for example, the PB1 protein set out in SEQ ID NO: 17).

[0024] In recombinant vaccinia viruses of the invention, the gene cassettes may be inserted in non-essential regions of the vaccinia virus genome, such as the deletion I region, the deletion II region, the deletion III region, the deletion IV region, the thymidine kinase locus, the D4R/5R intergenic region, or the HA locus. In exemplified embodiments of recombinant MVA, the insertion of the h1HA/M2e and h1HA gene cassettes is in the D4R/5R intergenic region and the insertion of the NP gene cassette is in the deletion III region. The recombinant MVA is derived from an MVA free of bovine spongiform encephalopathy (BSE) such as MVA74 LVD6 obtained from the National Institutes of Health.

[0025] The recombinant viruses of the invention may be formulated as pharmaceutical compositions according to methods known in the art. In some embodiments, the recombinant viruses are formulated as described in International Publication No. WO 2010/056991.

[0026] The invention provides methods of inducing a heterosubtypic influenza A immune response in an individual comprising administering compositions of recombinant vaccinia virus of the invention to the individual. In the methods, the composition may be administered as a single dose, a double dose or multiple doses. The administration route in humans may be inhalation, intranasally, orally, and parenterally. Examples of parenteral routes of administration include intradermal, intramuscular, intravenous, intraperitoneal and subcutaneous administration. The range of the human immunization dose may be about 10.sup.6 to about 10.sup.9 PFU. The methods of the invention induce humoral and cellular immune responses in the individual. Moreover, in embodiments of the invention the methods induce a protective immune response in the individual. The protective immune response may be where the individual exhibits no symptoms of infection, a reduction in symptoms, a reduction in virus titer in tissues or nasal secretions, and/or complete protection against infection by influenza virus.

[0027] The invention also provides kits for administering recombinant vaccinia virus of the invention packaged in a manner which facilitates their use to practice methods of the invention. In one embodiment, such a kit includes a recombinant virus or composition described herein, packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. Preferably, the recombinant virus or composition is packaged in a unit dosage form. The kit may further include a device suitable for administration according to a specific route of administration or for practicing a screening assay. Preferably, the kit contains a label that describes use of the recombinant vaccinia virus. In some embodiments, the kit comprises instructions for administration to a human subject.

[0028] Also provided are methods of producing a recombinant vaccinia virus expressing a gene cassette of the invention. As illustrated with MVA, the methods comprise the steps of: a) infecting primary chicken embryo cells or a suitable permanent cell line (e.g., avian) with MVA, b) transfecting the infected cells with a plasmid comprising the gene cassette and comprising DNA flanking the gene cassette that is homologous to a non-essential region of the MVA genome, c) growing the cells to allow the plasmid to recombine with the MVA genome during replication of the MVA in chicken cells thereby inserting the gene cassette into the MVA genome in the non-essential region, and d) obtaining the recombinant MVA produced. Exemplary chicken embryo cells are described in U.S. Pat. No. 5,391,491. (Slavik et al. 1983) Other avian cells (e.g., DF-1) are also contemplated. In the methods, the non-essential MVA region is the deletion I region, the deletion II region (Meyer et al. 1991), the deletion III region (Antoine et al. 1996), the deletion IV region (Meyer et al., supra; Antoine et al. 1998) the thymidine kinase locus (Mackett et al. 1982), the D4R/5R intergenic region (Holzer et al. 1998), or the HA locus (Antoine et al. supra). In one exemplified embodiment, the insertion is in the deletion III region. In another exemplified embodiment, the insertion is in the D4R/5R intergenic region. If two gene cassettes are to be inserted, the two are inserted in different non-essential regions. Gene cassettes may additionally be inserted into any other suitable genomic region or intergenomic regions.

[0029] Other vertebrate cell lines are useful for culture and growth of vaccinia virus of the invention. Exemplary vertebrate cells useful to culture vaccinia virus of the invention include, but are not limited to, MRC-5, MRC-9, CV-1 (African Green monkey), HEK (human embryonic kidney), PerC6 (human retinoblast), BHK-21 cells (baby hamster kidney), BSC (monkey kidney cell), LLC-MK2 (monkey kidney) and permanent avian cell lines such as DF-1.

[0030] Vero cells are an accepted cell line for production of viral vaccines according to the World Health Organization. In some embodiments, recombinant replicating vaccinia virus of the invention are produced in Vero cells.

[0031] Additional aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative rather than limiting.

FIGURES

[0032] FIG. 1 shows the amino acid sequence (SEQ ID NO: 15) of the headless HA protein encoded by recombinant MVA (rMVA) of the invention. The protein contains a signal sequence (grey), HA1 residues (red), a linker peptide of four glycines (black), HA1 residues (red), and the HA2 stalk region (black). Cysteines 58 and 63 and the polybasic cleavage site (amino acids 112-119) are underlined. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 14) of the headless HA protein encoded by rMVA of the invention.

[0033] FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) of headless HA/M2e fusion protein. The designed protein contains a signal sequence (grey), HA1 residues (red), a linker peptide of three glycines (black), the M2e of H5N1 (blue), the six amino acid linker GSAGSA (black), the M2e of H1N1 (equivalent to H2N2, H3N2; green), the six amino acid linker GSAGSA, the M2e of H9N2 (orange), the six amino acid linker GSAGSA, the M2e of H7N2 (pink), a linker peptide of three glycines (black), HA1 residues (red) and the HA2 stalk region (black). The polybasic cleavage site (amino acids 224-231) is underlined. FIG. 4 shows the nucleotide sequence (SEQ ID NO: 1) of the headless HA/M2e fusion protein encoded by rMVA of the invention.

[0034] FIG. 5 shows single-insert rMVAs containing influenza genes. A) indicates the h1HA, h1HA/M2e, M2, PB1, or M1 gene cassettes that are located in the recombinant MVA D4R/D5R intergenic locus, at the position corresponding to nucleotide 87,281 of wild type MVA (Antoine et al, supra). B) indicates the NP gene cassette is located in the del 111 locus at the position corresponding to nucleotide 142,992 of wild type MVA.

[0035] FIG. 6 shows a Western Blot of chicken cell lysates tested for influenza virus antigens. A) Expression of headless HA and the headless HA/M2e fusion protein using a detection antibody directed against HA. Lane 1, protein ladder, size in kDa; lane 2, MVA-h1HA; lane 3, MVA-h1HA/M2e; lane 4, MVA wt (negative control); and lane 5, MVA-HA-VN (positive control). B) Expression of the headless HA/M2e fusion protein using a detection antibody directed against M2. Lane 1, protein ladder, size in kDa; lane 2, MVA-M2-VN; lane 3, MVA-h1HA/M2e; and lane 4, MVA wt (negative control). The recombinant MVA-M2-VN expresses the M2 protein (weak band below 15 kDa). The anti-M2-antibody binds a peptide at the N-terminus of the M2 protein; thus the expression of the h1HA/M2e fusion protein is also detectable at around 70 kDa (lane 3).

[0036] FIG. 7 shows double-insert rMVAs containing influenza genes. The h1HA or h1HA/M2e gene cassette is located in the D4R/D5R intergenic locus, at the position corresponding to nucleotide 87,281 of wild type MVA. The NP gene cassette is located in the del 111 locus at the position corresponding to nucleotide 142,992 of the wild type MVA.

[0037] FIG. 8 shows a Western Blot of chicken cell lysates tested for influenza virus antigens. A) Expression of headless HA and the headless HA/M2e fusion protein using a detection antibody directed against HA. Lanes 1 and 7, protein ladder, size in kDa; lane 2, MVA-HA-VN (positive control); lane 3, MVA-h1HA; lane 4, MVA wt (negative control); lane 5, MVA-h1HA/M2e-NP; and lane 6, MVA-h1HA-NP. The h1HA/M2e fusion protein expressed by MVA-h1HA/M2e is visible at around 70 kDa (lane 5). The lower bands at around 40 kDa represent the h1HA expressed by MVA-h1HA-NP and MVA-h1HA. The control construct (MVA-HA-VN), expressing the full length HA protein express the HA0 (band around 80 kDa), the HA1 (band around 55 kDa, and the HA2 (band around 25 kDa). The expression of the HA2 protein is also visible in lanes 3, 5 and 6 as the h1HA and h1HA/M2e proteins also contain the polybasic cleavage site. The specific HA bands are absent in the negative control (lane 4). B) NP expression detected with an NP-specific antibody. Lane 1, protein ladder, size in kDa; lane 2, MVA-D3-NP-VN; lane 3, MVA-h1HA-NP; lane 4, MVA-h1HA/M2e-NP; and lane 5, MVA wt (negative control).

[0038] FIG. 9 shows monitoring of weight (A, B), clinical symptoms (C, D) and survival (E, F) after vaccination with recombinant MVAs and challenge with H5N1. As controls, mice were vaccinated with MVA-HA-VN, expressing the full-length HA of A/Vietnam/1203/2004, wt MVA or were treated with PBS (panels A, C, E). Mice were vaccinated with the single recombinant MVA-h1HA, MVA-h1HA/M2e, MVA-NP-VN or the double recombinants MVA-h1HA-NP and MVA-h1HA/M2e-NP (panels B, D, F). After challenge with wild-type H5N1, mice were monitored for 14 days.

[0039] FIG. 10 shows monitoring of weight (A, B), clinical symptoms (C, D) and survival (E, F) after vaccination with recombinant MVAs and challenge with H9N2 virus. As controls, mice were vaccinated with the whole virus preparation of H9N2, wt MVA or were treated with PBS (panels A, C, E). Mice were vaccinated with the single recombinant MVA-h1HA, MVA-h1HA/M2e, MVA-NP-VN or double recombinant MVA-h1HA-NP and MVA-h1HA/M2e-NP (panels B, D, F). After challenge with virulent mouse-adapted H9N2 influenza virus, mice were monitored for 14 days.

[0040] FIG. 11 shows triple-insert rMVAs containing influenza genes. The h1HA or h1HA/M2e and M1gene cassettes will be located in the D4R/D5R intergenic locus, at the position 87,281 nt of the wt MVA sequence. The NP gene cassette will be located in the del 111 locus at the position 142,992 nt of the wt MVA sequence.

EXAMPLES

[0041] The present invention is illustrated by the following examples wherein Example 1 describes the choice and design of influenza A antigens in exemplary recombinant MVA of the invention, Example 2 details the production of single-insert recombinant MVAs, Example 3 describes animal experiments with the single-insert MVAs, Example 4 details the production of double-insert recombinant MVAs, Example 5 describes animal experiments with the double-insert MVAs, Example 6 details the production of triple-insert recombinant MVAs and Example 7 describes animal experiments with the triple-insert MVAs.

Example 1

Choice and Design of Influenza a Antigens

[0042] Influenza headless HA, a headless HA/M2e fusion protein, NP, M1, M2 and PB1 were the influenza A antigens chosen to be encoded by exemplary recombinant MVA of the invention.

[0043] Monoclonal antibodies against the HA stalk domain, the HA2 region, are broadly cross-reactive and neutralize several subtypes of viruses (Ekiert et al. 2009; Kashyap et al. 2008; Okuno et al. 1993; Sanchez-Fauquier et al. 1987; Sui et al. 2009; Throsby et al. 2008). The antibodies target the HA2 region of the molecule and presumably act by preventing the conformational change of HA at low pH, thus presumably blocking fusion of viral and host membranes during influenza infection. However, the production of soluble, native (neutral pH-like) HA2 immunogen has proven to be difficult, owing to the metastable nature of HA (Chen et al. 1995). To induce an immune response against the neutral pH conformation, a headless HA was chosen as an antigen. The headless HA consists of two HA1 regions that interact with an HA2 subunit, stabilizing the neutral pH conformation (Bommakanti et al., supra; Steel et al., supra).

[0044] The extracellular domain of the M2 protein (M2e, 23AS) is highly conserved across influenza A virus subtypes. In animals, M2e specific antibodies reduce the severity of infection with a wide range of influenza A virus strains (Fan et al. 2004; Neirynck et al. 1999). Many groups have reported M2e-based vaccine candidates in different forms (De Filette et al. 2008; Denis et al. 2008; Eliasson et al. 2008; Fan et al., supra; Neirynck et al., supra). Recently, Zhao et al. reported that a tetra-branched multiple antigenic peptide vaccine based on H5N1 M2e induced strong immune responses and cross protection against different H5N1 clades and even heterosubtypic protection from 2009 H1N1 (Zhao et al. 2010b; Zhao et al. 2010a).

[0045] Vaccination using vectors expressing the conserved influenza NP, or a combination of NP and matrix protein has been studied in animal models and various degrees of protection against both homologous and heterologous viruses have been demonstrated (Price et al., supra; Ulmer et al. 1993). NP elicit a robust CD8.sup.+ T cell response in mice and in humans (McMichael et al., 1986; Yewdell et al., 1985) that, as epidemiological studies suggest, may contribute to resistance against severe disease following influenza A virus infection (Epstein 2006).

[0046] The headless HA included in rMVA of the invention is a new headless HA (h1HA) based on the VN/1203 influenza strain. The h1HA contains a polybasic cleavage site which is cleaved during expression from the rMVA exposing the fusion peptide for the immune system. The amino acid sequence of the h1HA is set out in FIG. 1 and in SEQ ID NO: 15. The nucleotide sequence of the MVA insert is set out FIG. 2 and SEQ ID NO: 14.

[0047] The amino acid sequence of the headless HA/M2e fusion protein included in rMVA of the invention is set out in FIG. 3 below and in SEQ ID NO: 2. The nucleotide sequence of the fusion protein is set out in FIG. 4 below and in SEQ ID NO: 1. In the fusion protein, the M2e domains of H5N1, H9N2, H7N2 and H1N1 (equivalent to H2N2, H3N2) form an M2e "head" on the h1HA. The four particular M2e domains were chosen to represent the M2e from seasonal and pandemic strains.

Example 2

Construction and Characterization of Single-Insert MVA Vectors

[0048] The following single-insert, recombinant MVA (rMVA) are utilized in the experiments described herein.

TABLE-US-00002 TABLE 2 rMVA Inserted influenza gene NCBI gene acc no. 1. MVA-h1HA headless HA based on AY818135 2. MVA-h1HA/M2e headless HA/M2e fusion based on AY818135 3. MVA-M1-VN Matrix protein 1 AY818144 4. MVA-M2-VN Matrix protein 2 EF541453 5. MVA-PB1-VN Polymerase subunit PB1 AY818129 6. MVA-mNP Nucleoprotein AY818138 7. Control MVA-HA-VN Hemagglutinin AY818135 8. Control MVA-wt No insert -- 9. Control PBS No insert --

[0049] For construction of single-insert rMVA vectors expressing h1HA, the h1HA/M2e fusion protein or PB1, the h1HA, h1HA/M2e and PB1 genes were chemically synthesized (Geneart, Inc., Regensburg, Germany). The synthetic genes are driven by the strong vaccinia early/late promoter mH5 (Wyatt et al. 1996) and terminated with a vaccinia virus specific stop signal downstream of the coding region that is absent internally. The gene cassettes were cloned in the plasmid pDM-D4R (Ricci et al., 2011) resulting in plasmids pDM-h1HA, pDM-h1HA/M2e and pDM-PB1-VN, respectively. The introduction of the foreign genes into the D4R/D5R intergenic region of MVA was done as described elsewhere (Ricci et al. 2011) resulting in viruses MVA-h1HA, MVA-h1HA/M2e, MVA-PB1-VN.

[0050] For the construction of the rMVA expressing M1, the M1 sequence (accession number AY818144) was placed downstream of the strong vaccinia early/late promoter selP (Chakrabarti et al. 1997) and cloned in pDM-D4R, resulting in pDM-M1-VN. The expression cassette of pDD4-M2-VN--including the M2 sequence (accession number EF541453) under the control of the mH5 promoter--was cloned in pDM-D4R resulting in pDM-M2-VN. The plasmids were then used for recombination with MVA according to Holzer et al, supra resulting in the viruses MVA-M1-VN and MVA-M2-VN, respectively as shown in FIG. 5A.

[0051] For the construction of single-insert MVAs expressing the NP protein, the NP expression cassette of pDD4-mH5-mNP-VN (Mayrhofer et al., supra) was cloned in plasmid pd3-lacZ-gpt, resulting in pd3-lacZ-mH5-NP-VN. Plasmid pd3-lacZ-gpt contains a lacZ/gpt selection marker cassette and a multiple cloning site (MCS) for insertion of genes of interest. The sequences are framed by genomic MVA sequences of the del III region. The marker cassette is destabilized by a tandem repeat of MVA del III flank, thus the final recombinant is free of any auxiliary sequences. The insertion plasmid directs the gene cassettes into the MVA deletion III (del III) region. After infection of primary chicken embryo cells with MOI 1, cells were transfected with pd3-lacZ-mH5-NP-VN according to the calcium phosphate technique (Graham and van der Eb 1973), resulting MVA-NP-VN shown in FIG. 5B. The MVA strain (MVA 1974/NIH clone 1) was kindly provided by B. Moss (National Institutes of Health). Recombinant virus is selected using the transient marker stabilization method (Scheiflinger et al, 1998).

[0052] The single-insert MVA vectors expressing the NP, PB1, M1, M2, h1HA, and h1HA/M2e were characterized by PCR and Western blot as described in Hessel et al, supra. Recombinant viruses were grown in CEC or DF-1 cells and purified by centrifugation through a sucrose cushion. Primary CEC were produced in-house and cultivated in Med199 (Gibco.RTM.) supplemented with 5% fetal calf serum (FCS). The DF-1 (CRL-12203) cell line was obtained from the ATCC (American Type Culture Collection) and cultivated in DMEM (Biochrom, Inc.) supplemented with 5% FCS.

[0053] The correct expression of the influenza proteins by the rMVAs was confirmed by Western blotting. For this purpose CEC or the permanent chicken cell line DF-1 were infected with a MOI of 0.1 and cell lysates were prepared 48-72 hrs post infections. The recombinant MVAs that express the h1HA (MVA-h1HA and MVA-h1HA/M2e) were analyzed in a Western blot using an anti-influenza A/Vietnam/1194/04 (H5N1) polyclonal serum (NIBSC 04/214) for detection. Donkey-anti-sheep alkaline phosphatase-conjugated IgG (Sigma Inc.) was used as a secondary antibody. The recombinant MVAs that express the M2 and M2e (MVA-M2-VN and MVA-h1HA/M2e) were analyzed in Western Blots using an anti-avian influenza M2 antibody binding a peptide present at the amino terminus of the H5N1 M2 (ProSci, Cat#4333). Goat-anti-rabbit alkaline phosphatase-conjugated IgG (Sigma Inc.) antibody was used as a secondary antibody. As shown in FIG. 6A, the recombinant MVAs expressing the h1HA (MVA-h1HA and MVA-h1HA/M2e) gene inserts induced expression of the HA containing antigens in avian DF-1 cells. The bands around 40 kDa in lane 2 represent the h1HA. The larger band at around 70 kDa in lane 3 represents the h1HA/M2e. The large band at around 80 kDa in lane 5 represents the HA0 hemagglutinin-precursor, which is cleaved into the HA1 and HA2 subunits represent by the bands at approximately 55 and 25 kDa. The specific h1HA, h1HA/M2e or HA bands are absent in the wild-type MVA control (lane 4).

[0054] FIG. 6B shows the M2 expression by MVA-M2-VN (lane 2) or MVA-h1HA/M2e (lane 3). The weak but specific band around 10 kDa in lane 2 represents the wild-type M2 protein whereas the larger band around 70 kDa represents the h1HA/M2e protein. Both bands are absent in the wild-type MVA control (lane 4).

[0055] The expression of the M1, NP and PB1 protein is detected with polyclonal guinea-pig anti-influenza H5N1 serum produced in house, a polyclonal goat antibody detecting the PB1 of Influenza A virus (Santa Cruz, Cat#: vC-19), and a monoclonal mouse-anti-NP-antibody (BioXcell, Cat# BE0159), respectively. The MVA-M1-VN and MVA-NP-VN induce expression of the M1 protein (around 27 kDa) and the NP protein (around 60 kDa) (not shown).

Example 3

Animal Experiments with the Single-Insert Vaccines

Protection Experiment

[0056] A standard protection experiment consists of two arms (primed with about 1.times.10.sup.3-1.times.10.sup.5 TCID.sub.50 H1N1v CA/07 and unprimed) of nine groups of mice each (respectively vaccinated i.m. with 1.times.10.sup.6 pfu of the nine vaccines and controls shown in Table 2), a group consisting of six animals resulting in 108 animals, defines one set. The animals of one set are challenged with one of the six challenge viruses shown in Table 3 below.

TABLE-US-00003 TABLE 3 Pre-treatment Challenge strain Subtype Abbreviation H1N1v/unprimed A/California/07/2009 H1N1 CA/07 H1N1v/unprimed A/Vietnam/1203/2004 H5N1 VN/1203 H1N1v/unprimed A/HongKong/G9/ H9N2 HK/G9 H1N1v/unprimed A/Victoria/210/2009 H3N2 VF09 H1N1v/unprimed A/FPV/Rostock/34 H7N1 RO/34 H1N1v/unprimed A/PR8/1934 H1N1 PR8

[0057] Female Balb/c mice are 8-10 weeks old at the pre-treatment time point and 14-16 weeks old at the time point of immunization with the vaccines and controls shown in Table 2. Mice were immunized intramuscularly twice (days 42 and 63) with 10.sup.6 pfu of the vaccines or wild type MVA, 3.75 .mu.g whole virus preparation H9N2 A/HongKong/G9/1997 or with buffer (PBS). At day 84, mice were challenged intranasally with 10.sup.3 TCID.sub.50 H5N1 A/Vietnam/1203/2004 (H5N1, CDC #2004706280), with 2.5.times.10.sup.4 TCID.sub.50 mouse adapted H9N2 A/HongKong/G9/1997 or with 1.66.times.10.sup.4 TCID.sub.50 H7N1 A/FPV/Rostock/34. The challenge doses correspond to approx. 30 LD50 for the H5N1 challenge and 32 LD50 for the H9N2 challenge per animal. Sera are collected at days 41, 62 and 85 and analyzed for HA-specific IgG concentration by HI titer or microneutralization assay.

[0058] The primary outcome of the animal experiments is protection as measured by lethal endpoint, weight loss, or lung titer. Further the ELISA titers of pooled pre-challenge sera measured against inactivated whole virus H5N1 strain A/Vietnam/1203/2004 are determined.

T Cell Experiments

[0059] Frequencies of influenza-specific CD4 and CD8 T cells are determined in immunized mice by flow cytometry. In a standard experiment, groups of 5 female BALB/c mice are immunized twice with the vaccines or controls listed in Table 2. Splenocytes are re-stimulated in-vitro using inactivated whole virus antigens of different influenza strains for CD4 T-cells and, when available, peptides representing the CD8 T-cell epitopes of the vaccine insert constructs and IFN-.gamma. production are measured. All experiments are performed twice, using a total of 140 animals.

Other Experiments

[0060] An evaluation of the cell-mediated immunity after a single immunization, demonstration of functional activity of cytotoxic T-cells in a VITAL assay and assessment of recruitment of influenza-specific T-cells into the lungs of challenged animals are also carried out. The induction/expansion of vaccine-specific T-cells is also monitored in the primed mouse model by immunizing mice which resolved a influenza virus infection once with these vaccines.

Example 4

Construction and Characterization of Double-Insert rMVA Vectors

[0061] The following double-insert, rMVA and controls are utilized in the experiments described herein.

TABLE-US-00004 TABLE 4 rMVA Inserted influenza gene Comment 1. MVA-h1HA-NP headless HA + NP Double insert construct 2. MVA-h1HA/M2e-NP headless HA/m2e fusion protein + NP Double insert construct 3. MVA-NP-VN nucleoprotein Control 4. MVA-HA-VN hemagglutinin Control 5. MVA-wt Empty vector Neg. control 6. PBS -- Neg. control

[0062] For the construction of the double insert rMVA vector co-expressing either the h1HA or h1HA/M2e gene cassette in combination with the NP protein gene cassette, the single insert MVA recombinants of Example 2 containing the h1HA or h1HA/M2e gene cassette are used. CEC cells were infected with MVA-h1HA or MVA-h1HA/Me2 and afterwards transfected with pd3-lacZ-mH5-NP-VN (see Example 2). Homologous recombination and propagation of the recombinant MVA vectors are performed as described in Example 2. The resulting double insert MVA vectors, named MVA-h1HA-NP or MVA-h1HA/M2e-NP, contain the h1HA or h1HA/M2e expression cassette in the D4R/D5R locus and the NP expression cassette in the del III locus. See FIG. 7.

[0063] The recombinant MVAs were characterized by Western Blot as described in Example 2. FIG. 8A shows the expression of the h1HA and h1HA/M2e after infection of CEC with MVA-h1HA-NP (lane 6) or MVA-h1HA/M2e-NP (lane 5). The bands around 40 kDa in lanes 3 and 6 represent the h1HA of the MVA-h1HA and MVA-h1HA-NP constructs. The band around 70 kDa in lane 5 represents the h1HA/M2e fusion protein. The HA bands are absent in the wild-type control in lane 4. The same samples were used for detection of NP protein expression in Western Blots (as described in Example 2). As shown in FIG. 8B, the recombinant MVAs MVA-h1HA-NP and MVA-h1HA/M2e-NP also induced expression of the NP protein in avian CEC cells. The bands around 60 kDa in lanes 2 to 4 represent the NP.

Example 5

Animal Experiments with the Double-Insert Vaccines or Vector Combinations

Protection Experiment

[0064] A standard experiment included eight groups of mice (vaccinated with the six vaccines and controls shown in Table 5) each group consisting of six animals. The protection experiments were carried out as described in Example 3. After challenge mice were monitored over a time period of 14 days and weight loss or symptoms including ruffled fur (score of 1), curved posture (score of 2), apathy (score of 3), and death (score of 4) were recorded. For ethical reasons, mice were euthanized after weight loss of .gtoreq.25%. Protection results are compiled in Table 5 and displayed in FIGS. 9 and 10.

TABLE-US-00005 TABLE 5 Protection of mice from death after double dose vaccinations with recombinant MVAs and homologous or heterologous challenge. VN1203.sup.(1) challenge HK/G9.sup.(2) After H5N1 Clinical Protection After H9N2 Clinical challenge n/nt Gr Vaccine score at day 14 n/nt.sup.(3) (%) score at day 14 (%) 1 MVA-h1HA-NP 2.83 2/6 (33) 0 6/6 (100) 2 MVA-h1HA/M2e-NP 1 5/6 (83) 0 6/6 (100) 3 MVA-h1HA 2.67 2/6 (33) 3.33 1/6 (17) 4 MVA-h1HA/M2e 4 0/6 (0) 2.67 2/6 (33) 5 MVA-NP-VN 3.33 2/6 (33) 0 6/6 (100) 6 Homologous 0 6/6 (100) 0 6/6 (100) control vaccine.sup.(4) 7 MVA-wt(5) 4 0/6 (0) 2.83 2/6 (33) 8 PBS 2.67 2/6 (33) 4 0/6 (0) .sup.(1)VN1203, challenge strain A/Vietnam/1203/2004; .sup.(2)HK/G9, challenge strain A/HongKong/G9/1997; .sup.(3)n/nt, survival per group, .sup.(4)Homologous control vaccine; (5)wild-type MVA (NIH74 LVD clone 6).

[0065] As positive control mice were vaccinated with homologous control constructs. In case of H5N1 challenge mice were vaccinated with MYA-HA-VN (Hessel et al., 2011) and in case of H9N2 challenge mice were vaccinated with an inactivated whole virus preparation of the H9N2 A/HongKong/G9/1997 influenza virus. Both controls induced full protection (Table 5; FIGS. 9 and 10, panels E). In the wild-type MVA and buffer groups all mice showed marked weight loss compared to the positive control groups and nearly all mice died after challenge. Mice vaccinated with the single recombinant MVAs (MVA-h1HA, MVA-h1HA/M2e, MVA-D3-NP-VN) showed no significantly better protection after the strong H5N1 challenge compared to the negative control groups (FIG. 9 A-F). Also against heterosubtypic (H9N2) challenge no significant protection was seen in MVA-h1HA and MVA-h1HA/M2e vaccinated groups (FIG. 10).

[0066] Surprisingly, however, vaccination with the double construct expressing the fusion protein h1HA/M2e and the NP protein resulted in nearly full protection (FIG. 9 B, D, F) after the H5N1 challenge with approx. 30 LD50 per animal. Also after heterosubtypic challenge (with approx. 32 LD50 H9N2 virus) mice were fully protected after vaccination with the double recombinant MVA-h1HA/M2e-NP. Furthermore, the double recombinant MVA-h1HA-NP and the single recombinant MVA-NP-VN induced full protection against the heterosubtypic challenge with H9N2 (FIG. 10, B, D, F). As can be seen in the weight monitoring (FIGS. 9 and 10, panels B) and in the clinical scores (FIGS. 9 and 10, panels D), the double construct MVA-h1HA/M2e-NP showed the best results presumably by combined beneficial effects contributed by the different influenza antigens.

T Cell Experiments

[0067] Frequencies of influenza-specific CD4 and CD8 T cells are determined in immunized mice by flow cytometry. In a standard protocol experiment, groups of 5 female BALB/c mice are immunized twice with the vaccines or controls listed in Table 4. Splenocytes are re-stimulated in-vitro using inactivated whole virus antigens of different influenza strains for CD4 T-cells and, when available, peptides representing the CD8 T-cell epitopes of the vaccine insert constructs and IFN-.gamma. production are measured. All experiments are performed twice.

Other Experiments

[0068] An evaluation of the cell mediated immunity after a single immunization, demonstration of functional activity of cytotoxic T-cells in a VITAL assay and assessment of recruitment of influenza-specific T-cells into the lungs of challenged animals are also carried out. The induction/expansion of vaccine-specific T-cells is also monitored in the primed mouse model by immunizing mice which resolved a influenza virus infection once with these vaccines.

Example 6

Construction and Characterization of Triple-Insert rMVA Vectors and Virus-Like Particles

[0069] Influenza virus-like particles (VLPs) induce humoral and cellular responses and can protect against lethal challenges (Bright et al. 2007; Pushko et al. 2005; Song et al. 2010). VLPs chosen for experiments herein comprise either h1HA or h1HA/M2e in combination with NP and M1. The VLPs are generated from triple-insert MVA vectors.

[0070] For the construction of the triple-insert MVA vectors co-expressing either h1HA or h1HA/M2e in combination with the M1 (SEQ ID NO: 11) and the NP protein (SEQ ID NO: 13), the M1 gene (SEQ ID NO: 10) of pDD4-M1-VN is cloned downstream of the synthetic early/late promotor selP (Chakrabarti et al. 1997). The resulting gene cassette is cloned downstream of the h1HA or h1HA/M2e gene cassette in pDM-h1HA or pDM-h1HA/M2e. The resulting plasmids harboring a double gene cassette (pDM-h1HA-M1 and pDM-h1HA/M2e-M1) are used for recombination into defective MVA as described above. Afterwards, a recombination with an NP gene cassette (SEQ ID NO: 12)-containing plasmid (pD3-lacZ-gpt-NP-VN) is done resulting in a triple-insert MVA virus. This triple-insert MVA is plaque purified under transient marker selection.

[0071] The triple-insert MVA vectors, named MVA-h1HA-M1-NP or MVA-h1HA/M2e-M1-NP contain the h1HA or h1HA/M2e expression cassette and M1 expression cassette in tandem order in the D4R/D5R locus and the NP expression cassette in the del III locus (FIG. 7).

[0072] Detection of VLPs is as follows. HeLa or 293 cells are seeded into T 175 cm.sup.2 flasks and grown in DMEM+10% FCS+Pen/Strep. To generate VLPs, cells are infected with 1 MOI of single-insert MVA or triple-insert MVA recombinants, respectively. Empty MVA vectors or single-insert MVA recombinants without M1 are used as controls. At 1 h post infection (p.i.), the medium is replaced by DMEM+Pen/Strep and culture medium is harvested 48 h p.i. and cellular debris is pelleted by centrifugation at 2.000.times.g for 10 min. The procedure for analyzing VLPs by sucrose gradient density flotation and sucrose cushion has been described previously (Chen et al. 2007; Chen et al. 2005; Gomez-Puertes et al. 2000). The samples are then analyzed by immunoblotting. Additionally, electron microscopy (EM) analysis with medium of infected cells is performed.

Example 7

Animal Experiments with the Triple-Insert Vaccines or Vector Combinations

[0073] A standard experiment includes 6 groups of primed and unprimed mice (vaccinated with the 6 vaccines and controls shown in Table 5), each group consisting of 6 animals, resulting in 36 animals (1 set). The animals are challenged with one of the 6 challenge viruses shown in Table 3. In sum, there are 6 sets of 72 animals each requiring 432 mice to assess cross-protection in the primed and naive models.

TABLE-US-00006 TABLE 5 rMVA Inserted influenza gene(s) comment 1. MVA-h1HA-M1-NP headless HA + nucleoprotein + 3 inserts matrix 1 2. MVA-h1HA/M2e-M1-NP headless HA/m2e fusion 3 inserts protein + nucleoprotein + matrix 1 3. MVA-tbd best construct from previous control screening 4. MVA-HA-VN hemagglutinin control 5. MVA-wt empty vector neg. control 6. PBS -- neg. control

[0074] The present invention is illustrated by the foregoing examples and variations thereof will be apparent to those skilled in the art. Therefore, no limitations other than those set out in the following claims should be placed on the invention.

[0075] All documents cited in this application are hereby incorporated by reference in their entirety for their disclosure described.

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[0116] Moss, B., M. W. Carroll, L. S. Wyatt, J. R. Bennink, V. M. Hirsch, S. Goldstein, W. R. Elkins, T. R. Fuerst, J. D. Lifson, M. Piatak, N. P. Restifo, W. Overwijk, R. Chamberlain, S. A. Rosenberg, and G. Sutter. 1996. Host range restricted, non-replicating vaccinia virus vectors as vaccine candidates. Adv Exp Med Biol 397:7-13. [0117] Neirynck, S., T. Deroo, X. Saelens, P. Vanlandschoot, W. M. Jou, and W. Fiers. 1999. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med 5:1157-63. [0118] Okuno, Y., Y. Isegawa, F. Sasao, and S. Ueda. 1993. A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol 67:2552-8. [0119] Poon, L. L., Y. H. Leung, J. M. Nicholls, P. Y. Perera, J. H. Lichy, M. Yamamoto, T. A. Waldmann, J. S. Peiris, and L. P. Perera. 2009. Vaccinia virus-based multivalent H5N1 avian influenza vaccines adjuvanted with IL-15 confer sterile cross-clade protection in mice. 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Sequence CWU 1

1

1911362DNAArtificial SequenceSynthetic nucleotide 1atggagaaaa tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt 120actgttacac atgcccaaga catactggaa aagaaacaca acgggaagct ctgcggagga 180ggaagtcttc taaccgaggt cgaaacgcct accagaaacg aatgggagtg cagatgcagc 240gattcaagtg atggaagtgc aggatcagcg agtcttctaa ccgaggtcga aacgcctatc 300agaaacgaat gggggtgcag atgcaacgat tcaagtgatg gaagtgcagg atcagcgagt 360cttctaaccg aggtcgaaac gcctaccaga aacggatggg agtgcaaatg cagcgattca 420agtgatggaa gtgcaggatc agcgagtctt ctaaccgagg tcgaaacgcc tatcagaaaa 480ggatgggagt gcaactgcag cgattcaagt gatggaggag gatgcaacac caagtgtcaa 540actccaatgg gggcgataaa ctctagcatg ccattccaca atatacaccc tctcaccatt 600ggggaatgcc ccaaatatgt gaaatcaaac agattagtcc ttgcgactgg gctcagaaat 660agccctcaaa gagagagaag aagaaaaaag agaggattat ttggagctat agcaggtttt 720atagagggag gatggcaggg aatggtagat ggttggtatg ggtaccacca tagcaatgag 780caggggagtg ggtacgctgc agacaaagaa tccactcaaa aggcaataga tggagtcacc 840aataaggtca actcgatcat tgacaaaatg aacactcagt ttgaggccgt tggaagggaa 900tttaacaact tagaaaggag aatagagaat ttaaacaaga agatggaaga cgggttccta 960gatgtctgga cttataatgc tgaacttctg gttctcatgg aaaatgagag aactctagac 1020tttcatgact caaatgtcaa gaacctttac gacaaggtcc gactacagct tagggataat 1080gcaaaggagc tgggtaacgg ttgtttcgag ttctatcata aatgtgataa tgaatgtatg 1140gaaagtgtaa gaaatggaac gtatgactac ccgcagtatt cagaagaagc gagactaaaa 1200agagaggaaa taagtggagt aaaattggaa tcaataggaa tttaccaaat actgtcaatt 1260tattctacag tggcgagttc cctagcactg gcaatcatgg tagctggtct atccttatgg 1320atgtgctcca atggatcgtt acaatgcaga atttgcattt aa 13622453PRTArtificial SequenceSynthetic peptide 2Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Lys His Asn Gly Lys Leu Cys Gly Gly Gly Ser Leu Leu 50 55 60 Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Cys Arg Cys Ser 65 70 75 80 Asp Ser Ser Asp Gly Ser Ala Gly Ser Ala Ser Leu Leu Thr Glu Val 85 90 95 Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser 100 105 110 Asp Gly Ser Ala Gly Ser Ala Ser Leu Leu Thr Glu Val Glu Thr Pro 115 120 125 Thr Arg Asn Gly Trp Glu Cys Lys Cys Ser Asp Ser Ser Asp Gly Ser 130 135 140 Ala Gly Ser Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Lys 145 150 155 160 Gly Trp Glu Cys Asn Cys Ser Asp Ser Ser Asp Gly Gly Gly Cys Asn 165 170 175 Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe 180 185 190 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys 195 200 205 Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Arg 210 215 220 Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 225 230 235 240 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His 245 250 255 His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr 260 265 270 Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 275 280 285 Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu 290 295 300 Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu 305 310 315 320 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu 325 330 335 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys 340 345 350 Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 355 360 365 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 370 375 380 Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys 385 390 395 400 Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gln 405 410 415 Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile 420 425 430 Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln 435 440 445 Cys Arg Ile Cys Ile 450 3568PRTInfluenza A virusMISC_FEATUREVN/1203 HA sequence 3Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90 95 Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile 340 345 350 Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355 360 365 Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe 405 410 415 Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430 Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 435 440 445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu 450 455 460 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505 510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly 515 520 525 Ile Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555 560 Ser Leu Gln Cys Arg Ile Cys Ile 565 43PRTArtificial SequenceSynthetic linker 4Gly Gly Gly 1 523PRTInfluenza A virusMISC_FEATUREH5N1 M2e sequence 5Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Cys 1 5 10 15 Arg Cys Ser Asp Ser Ser Asp 20 623PRTInfluenza A virusMISC_FEATUREH1N1 M2e sequence 6Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10 15 Arg Cys Asn Asp Ser Ser Asp 20 723PRTInfluenza A virusMISC_FEATUREH9N2 M2e sequence 7Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys 1 5 10 15 Lys Cys Ser Asp Ser Ser Asp 20 823PRTInfluenza A virusMISC_FEATUREH7N2 M2e sequence 8Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Lys Gly Trp Glu Cys 1 5 10 15 Asn Cys Ser Asp Ser Ser Asp 20 96PRTArtificial SequenceSynthetic linker 9Gly Ser Ala Gly Ser Ala 1 5 10986DNAInfluenza A virusmisc_featureM1 sequence 10atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccgtc aggccccctc 60aaagccgaga tcgcacagaa acttgaagat gtctttgcag gaaagaacac cgatctcgag 120gctctcatgg agtggctaaa gacaagacca atcctgtcac ctctgactaa agggattttg 180ggatttgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240cagaatgccc taaatggaaa tggagatcca aataatatgg atagggcagt taagctatat 300aagaagctga aaagagaaat aacattccat ggggctaagg aggtcgcact cagctactca 360accggtgcac ttgccagttg catgggtctc atatacaaca ggatgggaac ggtgactacg 420gaagtggctt ttggcctagt gtgtgccact tgtgagcaga ttgcagattc acagcatcgg 480tctcacagac agatggcaac tatcaccaac ccactaatca gacatgagaa cagaatggtg 540ctggccagca ctacagctaa ggctatggag cagatggcgg gatcaagtga gcaggcagcg 600gaagccatgg agatcgctaa tcaggctagg cagatggtgc aggcaatgag gacaattggg 660actcatccta actctagtgc tggtctgaga gataatcttc ttgaaaattt gcaggcctac 720cagaaacgaa tgggagtgca gatgcagcga ttcaagtgat cctattgttg ttgccgcaaa 780tatcattggg atcttgcact tgatattgtg gattcttgat cgtcttttct tcaaatgcat 840ttatcgtcgc cttaaatacg gtttgaaaag agggcctgct acggcagggg tacctgagtc 900tatgagggaa gagtaccggc aggaacagca gagtgctgtg gatgttgacg atggtcattt 960tgtcaacata gaattggagt aaaaaa 98611252PRTInfluenza A virusMISC_FEATUREM1 sequence 11Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Lys Leu Glu Asp Val Phe 20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95 Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Ala Thr Ile Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Ile Ala Asn Gln 195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Asn 210 215 220 Ser Ser Ala Gly Leu Arg Asp Asn Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys 245 250 121507DNAInfluenza A virusmisc_featureNP sequence 12atggcgtctc aaggcaccaa acgatcttat gaacagatgg aaactggtgg ggaacgccag 60aatgctactg agatcagggc atctgttgga agaatggtta gtggcattgg gaggttctac 120atacagatgt gcacagaact caaactcagt gactatgaag ggaggctgat ccagaacagc 180ataacaatag agagaatggt actctctgca tttgatgaaa gaaggaacag atacctggaa 240gaacacccca gtgcgggaaa ggacccgaag aagactggag gtccaattta tcggaggaga 300gacgggaaat gggtgagaga gctaattctg tacgacaaag aggagatcag gaggatttgg 360cgtcaagcga acaatggaga ggacgcaact gctggtctta cccacctgat gatatggcat 420tccaatctaa atgatgccac atatcagaga acgagagctc tcgtgcgtac tggaatggac 480ccaaggatgt gctctctgat gcaagggtca actctcccga ggagatctgg agctgccggt 540gcagcagtaa agggggtagg gacaatggtg atggagctga ttcggatgat aaaacgaggg 600atcaacgacc ggaatttctg gagaggcgaa aatggaagaa gaacaaggat tgcatatgag 660agaatgtgca acatcctcaa agggaaattc caaacagcag cacaaagagc aatgatggat 720caagtgcgag agagcagaaa tcctgggaat gctgaaattg aagatctcat ttttctggca 780cggtctgcac tcatcctgag aggatcagtg gcccataagt cctgcttgcc tgcttgtgtg 840tacggacttg cagtggccag tggatatgac tttgagagag aagggtactc tctggttgga 900atagatcctt tccgcctgct tcaaaacagc caggtcttta gtctcattag accaaatgag 960aatccagcac ataagagtca attagtgtgg atggcatgcc actctgcagc atttgaggac 1020cttagagtct caagtttcat cagagggaca agagtggtcc caagaggaca gctatccacc 1080agaggggttc aaattgcttc aaatgagaac atggaggcaa tggactccaa cactcttgaa 1140ctgagaagca gatattgggc tataagaacc agaagcggag gaaacaccaa ccagcagagg 1200gcatctgcag gacagatcag cgttcagccc actttctcgg tccagagaaa ccttcccttc 1260gaaagagcga ccattatggc agcatttaca ggaaatactg agggcagaac gtctgacatg 1320aggactgaaa tcataagaat gatggaaagt gccagaccag aagatgtgtc attccagggg 1380cggggagtct tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac 1440atgaataatg aaggatctta tttcttcgga gacaatgcag aggagtatga caattaaaga 1500aaaatac 150713498PRTInfluenza A virusMISC_FEATURENP sequence 13Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly 1 5 10 15 Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met 20 25 30 Val Ser Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45 Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu 50 55 60 Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu 65 70 75 80 Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile 85 90 95 Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp 100 105 110 Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp 115 120 125 Ala Thr Ala Gly Leu Thr His Leu Met Ile Trp His Ser Asn Leu Asn 130 135 140 Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175 Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190 Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg 195 200 205 Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn 210 215 220 Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp 225 230 235

240 Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu 245 250 255 Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His 260 265 270 Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly 275 280 285 Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe 290 295 300 Arg Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu 305 310 315 320 Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala 325 330 335 Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Val 340 345 350 Val Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn 355 360 365 Glu Asn Met Glu Ala Met Asp Ser Asn Thr Leu Glu Leu Arg Ser Arg 370 375 380 Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg 385 390 395 400 Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg 405 410 415 Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn 420 425 430 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met 435 440 445 Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe 450 455 460 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp 465 470 475 480 Met Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 495 Asp Asn 141026DNAArtificial SequenceSynthetic nucleotide 14atggagaaaa tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt 120actgttacac atgcccaaga catactggaa aagaaacaca acgggaagct ctgcggagga 180ggaggatgca acaccaagtg tcaaactcca atgggggcga taaactctag catgccattc 240cacaatatac accctctcac cattggggaa tgccccaaat atgtgaaatc aaacagatta 300gtccttgcga ctgggctcag aaatagccct caaagagaga gaagaagaaa aaagagagga 360ttatttggag ctatagcagg ttttatagag ggaggatggc agggaatggt agatggttgg 420tatgggtacc accatagcaa tgagcagggg agtgggtacg ctgcagacaa agaatccact 480caaaaggcaa tagatggagt caccaataag gtcaactcga tcattgacaa aatgaacact 540cagtttgagg ccgttggaag ggaatttaac aacttagaaa ggagaataga gaatttaaac 600aagaagatgg aagacgggtt cctagatgtc tggacttata atgctgaact tctggttctc 660atggaaaatg agagaactct agactttcat gactcaaatg tcaagaacct ttacgacaag 720gtccgactac agcttaggga taatgcaaag gagctgggta acggttgttt cgagttctat 780cataaatgtg ataatgaatg tatggaaagt gtaagaaatg gaacgtatga ctacccgcag 840tattcagaag aagcgagact aaaaagagag gaaataagtg gagtaaaatt ggaatcaata 900ggaatttacc aaatactgtc aatttattct acagtggcga gttccctagc actggcaatc 960atggtagctg gtctatcctt atggatgtgc tccaatggat cgttacaatg cagaatttgc 1020atttaa 102615341PRTArtificial SequenceSynthetic peptide 15Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Lys His Asn Gly Lys Leu Cys Gly Gly Gly Gly Cys Asn 50 55 60 Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe 65 70 75 80 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys 85 90 95 Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Arg 100 105 110 Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 115 120 125 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His 130 135 140 His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr 145 150 155 160 Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 165 170 175 Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu 180 185 190 Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu 195 200 205 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu 210 215 220 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys 225 230 235 240 Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 245 250 255 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 260 265 270 Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys 275 280 285 Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gln 290 295 300 Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile 305 310 315 320 Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln 325 330 335 Cys Arg Ile Cys Ile 340 162274DNAInfluenza A virusmisc_featurePB1 sequence 16atggatgtca atccgacttt acttttcttg aaagtaccag tgcaaaatgc tataagtacc 60accttccctt atactggaga ccctccatac agccatggaa cagggacagg atacaccatg 120gacacagtca acagaacaca ccaatattca gaaaagggga agtggacaac aaacacagag 180actggagcac cccaactcaa cccgattgat ggaccactac ctgaggataa tgagcccagt 240gggtacgcac aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccaccca 300gggatctttg aaaactcgtg tcttgaaacg atggaaattg ttcaacaaac aagagtggat 360aaactgaccc aaggtcgcca gacctatgac tggacattga atagaaacca accggctgca 420actgctttgg ccaacactat agaaatcttc agatcgaacg gtctaacagc caatgaatcg 480ggacggctaa tagatttcct caaggatgtg atggagtcaa tggataagga agaaatggag 540ataacaacac atttccagag aaagagaagg gtgagggaca acatgaccaa gaaaatggtc 600acacaaagaa caatagggaa gaaaaaacaa aggctgaaca aaaagagcta cctgataaga 660gcactgacac tgaacacaat gacaaaagat gcagaaagag gcaaattgaa gaggcgagcg 720attgcaacac ccggaatgca aatcagagga ttcgtgtact ttgttgaaac actagcgagg 780agtatctgtg agaaacttga gcaatctgga ctcccagtcg gagggaatga gaagaaggct 840aaattggcaa acgtcgtgag gaagatgatg actaactcac aagatactga actctccttt 900acaattactg gagacaatac caaatggaat gagaatcaga atcctaggat gtttctggca 960atgataacgt acatcacaag gaaccagcca gaatggtttc ggaatgtctt aagcatagct 1020cctataatgt tctcaaacaa aatggcgaga ctaggaaaag gatacatgtt cgaaagtaag 1080agcatgaagt tacgaacaca aataccagca gaaatgcttg caaacattga tcttaaatac 1140ttcaatgaat taacgaaaaa gaaaattgag aaaataaggc ctctattaat agatggtaca 1200gcctcattga gccctggaat gatgatgggc atgttcaaca tgctgagtac agtcctagga 1260gtttcaatcc tgaatcttgg acagaaaagg tacaccaaaa ccacatattg gtgggacgga 1320ctccaatcct ctgatgattt cgctctcatc gtaaatgcac cgaatcatga gggaatacaa 1380gcaggagtgg ataggtttta taggacttgt aaactagttg gaatcaatat gagcaagaag 1440aagtcttaca taaatcggac agggacattt gaattcacga gctttttcta ccgctatgga 1500tttgtagcca atttcagtat ggagctgccc agttttggag tgtctggaat taatgaatcg 1560gccgacatga gcattggtgt tacagtgata aaaaacaata tgataaacaa cgaccttggg 1620ccagcaacag ctcagatggc tcttcagtta ttcatcaagg actacagata cacataccga 1680tgccacagag gggatacgca aatccaaaca aggagatcat tcgagctgaa gaagctgtgg 1740gagcaaaccc gttcaaaggc aggactgttg gtttcagatg gaggaccaaa tctatacaat 1800atccgaaacc tccatattcc tgaagtctgc ttaaaatggg aattgatgga tgaagattac 1860cagggcagac tgtgtaatcc tctgaatcca ttcgtcagcc ataaggaaat tgaatctgtc 1920aacaatgctg tagtaatgcc agctcatggc ccggccaaga gtatggaata tgatgccgtt 1980gcaactacac attcatggat tcctaaaagg aaccgttcca ttctcaatac gagtcaaagg 2040ggaattcttg aggatgaaca gatgtaccag aagtgctgca atctattcga gaaattcttc 2100cccagcagtt catatcggag gccagttgga atttccagca tggtggaggc catggtgtct 2160agggcccgaa ttgacgcacg aatcgatttc gagtctggaa ggattaagaa agaagagttt 2220gccgagatca tgaagatctg ttccaccatt gaagaactca gacggcaaaa atag 227417757PRTInfluenza A virusMISC_FEATUREPB1 sequence 17Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Val Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Ile Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Ile Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser 145 150 155 160 Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Asp Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Lys Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Asn Ile Asp Leu Lys Tyr Phe Asn Glu Leu 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr 385 390 395 400 Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser 405 410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr Thr 420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445 Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510 Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr 515 520 525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu 610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val 625 630 635 640 Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650 655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg 660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met 675 680 685 Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 Lys Glu Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750 Leu Arg Arg Gln Lys 755 1899DNAArtificial SequencePromoter 18aaaaattgaa aataaataca aaggttcttg agggttgtgt taaattgaaa gcgagaaata 60atcataaata atttcattat cgcgatatcc gttaagttt 991939DNAArtificial SequenceSynthetic promoter 19aaaaattgaa attttatttt ttttttttgg aatataaat 39

Claims

1. A recombinant vaccinia virus comprising a gene cassette encoding a fusion protein comprising at least one influenza A M2 extracellular domain (M2e) polypeptide inserted in an influenza A headless hemagglutinin (h1HA) polypeptide.

2. The recombinant vaccinia virus of claim 1 wherein the fusion protein comprises one selected from the group consisting of i) the h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO: 2, ii) the VN/1203 HA amino acid sequence set out in SEQ ID NO: 3, and iii) HA1 amino acids 17-58 of SEQ ID NO: 3, a peptide linker, at least one M2e polypeptide, a peptide linker, HA1 amino acids 290-343 of SEQ ID NO: 3 and HA2 amino acids 344-568 of SEQ ID NO: 3.

3-4. (canceled)

5. The recombinant vaccinia virus of claim 2, wherein the peptide linkers linking the HA amino acids and M2e amino acids comprise the amino acids GGG set out in SEQ ID NO: 4.

6. The recombinant vaccinia virus of claim 1, wherein the M2e polypeptide comprises one selected from the group consisting of i) the H5N1 M2e amino acid sequence set out in SEQ ID NO: 5, ii) the H1N1 M2e amino acid sequence set out in SEQ ID NO: 6, iii) the H9N2 M2e amino acid sequence set out in SEQ ID NO: 7, and iv) the H7N2 M2e amino acid sequence set out in SEQ ID NO: 8.

7-9. (canceled)

10. The recombinant vaccinia virus of claim 1, wherein the fusion protein comprises more than one M2e polypeptide and the M2e polypeptides are linked by a peptide linker.

11. The recombinant vaccinia virus of claim 10, wherein the peptide linker linking the M2e polypeptides comprises the amino acids GSAGSA set out in SEQ ID NO: 9.

12. The recombinant vaccinia virus of claim 1, wherein expression of the h1HA/M2e fusion protein from the gene cassette is under the control of an mH5 promoter or a selP promoter.

13. (canceled)

14. A recombinant vaccinia virus comprising the gene cassette set out in SEQ ID NO: 1.

15. The recombinant vaccinia virus of claim 1, further comprising a gene cassette encoding an influenza A matrix protein 1 (M1) and a gene cassette encoding an influenza A nucleoprotein (NP).

16-23. (canceled)

24. A pharmaceutical composition comprising the recombinant vaccinia virus of claim 1.

25-26. (canceled)

27. A method of inducing a heterosubtypic immune response to influenza A virus in an individual comprising administering a pharmaceutical composition comprising the recombinant vaccinia virus of claim 1 to the individual.

28-31. (canceled)

32. A recombinant vaccinia virus comprising a first gene cassette encoding a fusion protein comprising at least one influenza A M2 extracellular domain (M2e) polypeptide inserted in an influenza A headless hemagglutinin (h1HA) polypeptide and a second gene cassette encoding influenza A nucleoprotein (NP).

33. The recombinant vaccinia virus of claim 32, wherein the fusion protein comprises one selected from the group consisting of i) the h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO: 2, ii) the VN/1203 HA amino acid sequence set out in SEQ ID NO: 3, and iii) HA1 amino acids 17-58 of SEQ ID NO: 3, a peptide linker, at least one M2e polypeptide, a peptide linker, HA1 amino acids 290-343 of SEQ ID NO: 3 and HA2 amino acids 344-568 of SEQ ID NO: 3.

34-35. (canceled)

36. The recombinant vaccinia virus of claim 33, wherein the peptide linkers linking the HA amino acids and M2e amino acids comprise the amino acids GGG set out in SEQ ID NO: 4.

37. The recombinant vaccinia virus of claim 32, wherein the M2e polypeptide comprises one selected from the group consisting of i) the H5N1 M2e amino acid sequence set out in SEQ ID NO: 5, ii) the H1N1 M2e amino acid sequence set out in SEQ ID NO: 6, iii) the H9N2 M2e amino acid sequence set out in SEQ ID NO: 7, and iv) the H7N2 M2e amino acid sequence set out in SEQ ID NO: 8.

38-43. (canceled)

44. The recombinant vaccinia virus of claim 32, wherein expression of the h1HA/M2e fusion protein from the first gene cassette is under the control of an mH5 promoter or a selP promoter.

45-46. (canceled)

47. The recombinant vaccinia virus of claim 32, wherein expression of NP from the second gene cassette is under the control of an mH5 promoter or selP promoter.

48-59. (canceled)

60. A pharmaceutical composition comprising the recombinant vaccinia virus of claim 1.

61-62. (canceled)

63. A method of inducing a heterosubtypic immune response to influenza A viruses in an individual comprising administering a pharmaceutical composition comprising the recombinant vaccinia virus of claim 32 to the individual.

64. A pharmaceutical composition for use in inducing a heterosubtypic immune response to influenza A viruses in an individual, the method comprising the step of administering a pharmaceutical composition comprising the recombinant vaccinia virus of claim 32 to the individual.

65-101. (canceled)