WO2010144797A2

WO2010144797A2 - Influenza vaccines with enhanced immunogenicity and uses thereof

Info

Publication number: WO2010144797A2
Application number: PCT/US2010/038309
Authority: WO
Inventors: Yichen Lu; Neal Touzjian; Shu Li
Original assignee: Vaccine Technologies, Incorporated
Priority date: 2009-06-12
Filing date: 2010-06-11
Publication date: 2010-12-16
Also published as: WO2010144797A3

Abstract

The present invention generally relates to Influenza vaccine compositions and methods of use. One aspect of the present invention relates to a vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide, and optionally an influenza virus matrix (M) protein. Another aspect of the present invention relates to an influenza virus vaccine composition and methods of use, comprising an influenza virus NP and/or M protein and a Bacillus anthracis lethal factor (LF) polypeptide, such as a LFn polypeptide, a N-terminal portion of the LF polypeptide. In some embodiments, the LF polypeptide can be fused or otherwise associated with the NP or M polypeptide, or alternatively, or in addition, associated with any or all of the influenza virus antigen polypeptides (e. g. HA, NA, etc) in the vaccine composition.

Description

FIELD OF THE INVENTION

[0001] The present application is generally directed to compositions and methods for vaccinations against influenza virus, and in particular delivering an exogenous influenza virus protein to the cytosol of a cell, and methods and thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This Application claims benefit under 35 U.S.C. §119(e) to U.S. Provisional Application 61/186,422, filed, June 12, 2009 the entire contents of which is incorporated herein by reference.

BACKGROUND OF INVENTION

[0003] Influenza (flu) is an acute infection of the bronchial tubes and is caused by the influenza virus. Flu is highly contagious and causes people to feel severely ill. An average of 5% of the world's population is annually infected with this virus. Typically, in a year's normal two flu seasons (one per hemisphere), there are between three and five million cases of severe illness and up to 500,000 deaths worldwide, which by some definitions is a yearly influenza epidemic. Although the incidence of influenza can vary widely between years, approximately 36,000 deaths and more than 200,000 hospitalizations are directly associated with influenza every year in America. Every ten to twenty years, a pandemic occurs, which infects a large proportion of the world's population and can kill tens of millions of people. Three pandemics occurred in the previous century, the first one, the "Spanish Flu" in 1918- 1919, was responsible for at least 50 million human deaths worldwide.

[0004] Vaccines can provide protection against infection by the influenza viruses. The most common human vaccine is the trivalent influenza vaccine that contains purified and inactivated materials from three viral strains, usually inactivated whole influenza virus vaccine or live attenuated influenza virus vaccine. Typically, these vaccines include materials from two influenza A virus subtypes and one influenza B virus strain. A vaccine formulated for one year may be ineffective in the following year, since the influenza virus changes their coat proteins, haemagglutinin (HA) and neuraminidase (N), rapidly over time, and different strains become dominant. As such, a yearly vaccination is recommended. [0005] Currently, the four FDA-approved Influenza virus vaccines in the Untied States are Sanofi- Aventis' FLUZONE^®, Chiron's FLUVIRIN^®, GlaxoSmithKline's FLUARIX^®, and Medlmmune's FLUMIST^®. The Sanofi-Aventis, Chiron and GlaxoSmithKline vaccines are trivalent, inactivated virus vaccines containing both Types A and B Influenza viruses. Medlmmune vaccine contain attenuated live Influenza virus for an intranasal application.

[0006] These traditional influenza vaccines are manufactured using three potentially threatening strains of flu virus. These are usually grown in fertilized chicken eggs. The manufacturing process of viruses is long, beginning with the supply of embryonated eggs, hundreds of millions in number, in which the flu viruses are cultivated, through the different steps of vaccine production — egg harvest, purification, inactivation, splitting the virus down to the final vaccine formulation and aseptic filling in the appropriate containers. With the egg method, each of the three targeted flu strains for a given year is injected into separate fertilized eggs, where the viruses multiply. The three viruses are later harvested from the eggs, purified, and killed. Using a detergent, manufacturers "split" the viruses, releasing the surface antigens HA and NA to increase accessibility to the immune cells of the body. Finally, the three split viruses are combined to make one dose of flu vaccine. In usual times, such a production cycle takes over 70 weeks. In the event of a major influenza epidemic, the availability of a potent and safe vaccine would be a major concern. Moreover, these types of flu vaccines are contraindicated for those with severe allergies to egg proteins and people with a history of Guillain-Barre syndrome. Further, there are risks associated with impurities in eggs, such as antibiotics and other viruses, which may cause sterility problems. [0007] The Sanofi-Aventis, Chiron and GlaxoSmithKline vaccines are sterile suspensions prepared from influenza viruses propagated in embryonated chicken eggs and are for intramuscular administration. Influenza virus types A and B are contained in the vaccine. The 2008-2009 season vaccine contains A/Brisbane/59/2007 (HlNl)-like virus, A/Brisbane/10/2007 (H3N2)-like virus, and B/Florida/4/2006- like virus. Each of the influenza viruses is produced and purified separately. After harvesting the virus- containing fluids, each influenza virus is concentrated and purified by zonal centrifugation using a linear sucrose density gradient solution containing detergent to disrupt the viruses. Following dilution, the vaccine is further purified by diafiltration. Each influenza virus solution is inactivated with formaldehyde (Sanofi-Aventis and GlaxoSmithKline) or with betapropiolactone (Chiron). The virus is then chemically disrupted using a nonionic surfactant, octoxinol-9 (TRITON^® X-100) or sodium deoxycholate, producing a "split virus." The split virus is then further purified by chemical means. Each split inactivated virus is then suspended in sodium phosphate-buffered isotonic sodium chloride solution. The vaccine is formulated from the 3 split inactivated virus solutions.

[0008] Medlmmune's FLUMIST^® is a newly licensed live attenuated vaccine that is administered by nasal spray to patients between the ages of 5 and 49. This new vaccine is not licensed for use in "at-risk" populations. Medlmmune produced approximately 4 million doses of FLUMIST^® vaccine for the 2003 flu season. This vaccine is also grown on embryonated chicken eggs. This vaccine is a live attenuated formulation that is delivered by nasal spray. In addition to limitations in the amount of doses that can be manufactured each year, the vaccine is not licensed for use in the young and elderly populations, which need protection from influenza the most.

[0009] For these reasons, alternative approaches are being developed: live vectored vaccines, plasmid DNA vaccines, recombinant influenza antigens, synthetic peptides or specific adjuvants. Live vectored vaccines are good at inducing a strong cellular response, but pre-existing (e.g. adenovirus) or vaccine- induced immunity against the vector can jeopardize the efficiency of additional vaccine dose (Casimiro et al, 2003, J. Virol., 77:6305-6313). Plasmid DNA vaccines also can induce a cellular response (Casimiro et al, 2003, J. Virol., 77:6305-6313) but it remains weak in humans (Mc Conkey et al, 2003, Nature Medicine, 9:729-735,) and the antibody response is very poor. In addition, synthetic peptides are currently being evaluated in clinical trials (Khong et al, 2004, J Immunother. 27:472-477), but the efficacy of such vaccines encoding a limited number of T cell epitopes may be hampered by the appearance of vaccine escape mutants or by the necessity of first selecting for HLA-matched patients.

SUMMARY OF THE INVENTION

[0010] The present invention provides improvements to any of the "standard" or "common" Influenza vaccine compositions. In one embodiment, provided herein is a vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide. In another embodiment, provided herein is a vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide and an influenza virus matrix (M) protein. Addition of the NP increased immunogenicity of the vaccine.

[0011] In another aspect, the present invention also provides a "standard" or "common" influenza virus vaccine composition, further comprising an influenza virus NP and/or M protein and a Bacillus anthracis letal factor (LF) polypeptide. In one embodiment, the LF polypeptide is an LFn polypeptide, a N-terminal portion of the LF polypeptide. The LF polypeptide can be fused or otherwise associated with the NP or M polypeptide, or alternatively, or in addition, associated with any or all of the influenza virus antigen polypeptides (e. g. HA, NA, etc) in the vaccine composition.

[0012] Provided herein also is a method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising an influenza virus NP polypeptide or a preparation comprising an influenza virus NP polypeptide and an influenza virus matrix (M) protein.

[0013] In some aspects, the influenza virus polypeptide, the LF polypeptide, or the LF fusion polypeptide are expressed and isolated from a bacculovirus expression system.

[0014] The vaccine composition described herein, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD₅₀ of a H5N2 influenza virus or an influenza virus having same HA/NA serotypes as the source of influenza virus-derived components in the vaccine composition. [0015] In one embodiment, the antigen consists of the influenza virus NP polypeptide of SEQ. ID. No.

6, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[0016] In one embodiment, the antigen consists of the influenza virus Ml polypeptide of SEQ. ID. No.

7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[0017] In one embodiment, the antigen consists of the influenza virus M2 polypeptide of SEQ. ID. No.

8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[0018] The vaccine composition described herein can further comprise an adjuvant. The adjuvant can be selected from the group consisting of QS- 21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL- 1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59. [0019] In some embodiments, in the vaccine composition described herein, LF polypeptide is an N- terminal (LFn) polypeptide, or conservative substitution variant thereof, that promotes transmembrane delivery to the cytosol of an intact cell. In other embodiments, the LFn polypeptide is N-glycosylated.

[0020] In other embodiments, LFn polypeptide comprises at least the 60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

[0021] In other embodiments, LFn polypeptide comprises at least the 80 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

[0022] In other embodiments, LFn polypeptide comprises at least the 104 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

[0023] In other embodiments, LFn polypeptide comprises the amino acid sequence corresponding to

SEQ. ID. No. 5, or a conservative substitution variant thereof.

[0024] In other embodiments, LFn polypeptide does not bind B. anthracis protective antigen protein.

[0025] In other embodiments, LFn polypeptide substantially lacks the amino acids 1-33 of SEQ. ID.

No. 3.

[0026] In other embodiments, LFn polypeptide consists of SEQ. ID. No. 5, or a conservative substitution variant thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Described herein is an improvement on influenza vaccines that can be used on its own or in conjunction or admixture with essentially any of the existing influenza vaccine approaches. [0028] In one aspect, described herein is any of the "standard" or "common" vaccine compositions, plus an influenza virus nucleoprotein (NP) polypeptide, or together with an influenza virus matrix protein (M).

[0029] In another aspect, described herein is a "standard" or "common" influenza virus vaccine composition, further comprising an influenza virus NP and/or M protein and a Bacillus anthracis letal factor (LF) polypeptide. In one embodiment, the LF polypeptide is an LFn polypeptide as the term is defined herein. The LF polypeptide can be fused or otherwise associated with the NP or M polypeptide, or alternatively, or in addition, associated with any or all of the influenza virus antigen polypeptides (e. g. HA, NA, etc) in the vaccine composition.

[0030] The "standard" or "common" influenza vaccines can be divided into two major categories: (1) vaccines comprising virus(es) cultured in living cells and (2) vaccines comprising viral proteins or viral subunits. The viral proteins can be subdivided into those purified from viruses or recombinantly expressed, fused or not-fused to non-influenza proteins, fragments, peptides, mutants, variants etc.

Vaccine compositions comprising cultured viruses

[0031] The inventors have discovered that the combination of an isolated recombinant baculovirus- expressed influenza virus nucleoprotein (NP) polypeptide with inactivated or attenuated influenza virus greatly enhanced the induced immune response and antibody production produced compared to the induced immune response and antibody production by the inactivated or attenuated virus alone without the baculovirus-expressed NP. The influenza virus is first cultured, harvested and then inactivated or attenuated before use. In addition, the inventors found that the combination of isolated recombinant baculovirus-expressed influenza virus NP and isolated recombinant baculovirus-expressed influenza virus matrix (M) proteins with inactivated or attenuated virus significantly enhanced the induced immune response and antibody production. The M protein can be an influenza virus Ml or M2 protein. Accordingly, isolated recombinantly expressed influenza virus nucleoprotein (NP) and matrix (M) proteins can be useful in vaccine preparations for immunization protection against influenza infections. [0032] Accordingly, embodiments of the invention provide vaccine compositions that have improved immungencity, comprising a cultured, inactivated or attenuated influenza virus and an isolated influenza virus nucleoprotein (NP) polypeptide.

[0033] Additionally, embodiments of the invention provide vaccine compositions that have improved immungencity, comprising a cultured, inactivated or attenuated influenza virus, an isolated influenza virus matrix (M) protein and an isolated influenza virus nucleoprotein (NP) polypeptide. [0034] In one embodiment, the isolated influenza virus matrix (M) protein is an Ml protein. [0035] In another embodiment, the isolated influenza virus matrix (M) protein is an M2 protein. [0036] In yet another embodiment, the isolated influenza virus matrix (M) protein is an M2e protein. In additional embodiments, various combinations of isolated M proteins can be included in the vaccine compositions.

[0037] Accordingly, in one embodiment, provided are vaccine compositions that have improved immungencity, comprising a cultured, inactivated or attenuated influenza virus, an isolated influenza virus Ml protein and an isolated influenza virus nucleoprotein (NP) polypeptide.

[0038] In another embodiment, provided are vaccine compositions that have improved immungencity, comprising a cultured, inactivated or attenuated influenza virus, an isolated influenza virus M2 protein and an isolated influenza virus nucleoprotein (NP) polypeptide.

[0039] In another embodiment, provided are vaccine compositions that have improved immungencity, comprising a cultured, inactivated or attenuated influenza virus, an isolated influenza virus Ml protein, an isolated influenza virus M2 protein, and an isolated influenza virus nucleoprotein (NP) polypeptide. [0040] In another embodiment, provided are vaccine compositions that have improved immungencity, comprising a cultured, inactivated or attenuated influenza virus, an isolated influenza virus Ml protein, an isolated influenza virus M2e protein, and an isolated influenza virus nucleoprotein (NP) polypeptide, which can be useful in vaccine preparations for immunization against influenza infections. [0041] In some embodiments, the isolated influenza virus Ml, M2, M2e and NP are fusion polypeptides. In a preferred embodiment, the isolated influenza virus Ml, M2, M2e and NP are B. anthracis Lethal Factor (LF) fusion polypeptides, i. e. influenza virus Ml, M2, M2e or NP fused to the B. anthracis LF protein. In another preferred embodiment, the isolated influenza virus Ml, M2, M2e and NP are B. anthracis N-terminal Lethal Factor (LFn) fusion polypeptides, i. e. influenza virus Ml, M2, M2e or NP fused to the B. anthracis LFn protein. The influenza virus Ml, M2, M2e and NP can also be fused to fragments of the B. anthracis LF protein, fragments that are at least 15 amino acids residues in length. The fusion is via a peptide bond between the viral protein and the LF protein or fragment. [0042] In one embodiment, the immungencity induced includes either innate or acquired immunity or both innate and acquired immunity.

[0043] In one embodiment, the cultured influenza virus is selected from the group consisting of Influenza virus A, Influenza virus B, and Influenza virus C. These three species infect humans. These are RNA viruses of the family Orthomyxoviridae . The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The Influenza A virus can be subdivided into different serotypes based on the antibody response to the hemagglutinin (HA), neuraminidase (NA) glycoproteins on the outside of the viral particles, forming the basis of the H and N distinctions in, for example, H5N1. Influenza B almost exclusively infects humans and is less common than influenza A.

[0044] In some embodiments of the vaccine compositions described herein, the cultured influenza virus includes but is not limited to one or more of Influenza virus A HlNl A/Sw/Iowa/31 , H2N3 A/Mal/Al/77, H2N9 A/Pintail/Alb/293/77, H3N2 A/Ty/Eng/69, H3N8 A/Dk/Ukraine/1/63, H4N8 A/Dk/Czech/56, H5N1 A/Ck/Scot/59, H5N2 A/Quail/Ore/20719/86, H5N2 A/Ck/Wa/13413/84, H5N2 A/Ck/Penn/13701/83, H5N2 A/Ty/Min/3689-1551/81, H5N3 A/Ty/cA/35621/84, H5N3 A/Tern/SA/61, H5N8 A/Ty/Ireland/83, H5N9 A/Ty/Wis/68, H5N9 A/Ty/Ont/7732/66, H5N9 A/Ck/Que/14588-19 (Mex. Isolate), H5N2 A/Ck/Hidalgo/26654-1368/94 (Mex. Isolate), H5N2 A/Ck/Pue/8623-607 (Mex. Isolate), H5N? A/Emu/Tx/39924/93, H5N3 A/Emu/Tx/39924/93 (IB clone E2), H6N2 Field Isolate, Cnn00053, H6N8 A/Ty/Ont/63, H7N2 A/Ty/Ore/71, H7N3 A/Ck/Aust/3634/92, H7N3 A/Ty/MN/29206/83, H7N7 A/Ck/Vic/32972/85, H7N8 A/Magrob/China/28710/93, H7N9 A/Ty/MN/38429/88, H8N4 A/Ty/Ont/61181/67, H9N2 A/Ty/MN/12877/1285/81, H9N2 A/Ty/Wis/1/66, H9N9 A/Pheasent/Wa/37, H10N7 A/Ck/Ger many/49, H10N8 A/Quail/Ithaca/1117/65, HI lNl A/Dk/Eng/56, H11N9 A/Dk/Memphis/546/74, H12N1 A/Dk/Alberta/60/76, H13N1 A/Gull/MD/704/77, H14N5 A/Mal/Gurjev/263/83, Hl, H1N2, , influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, B/Shanghai, B/Paris, and B/Hong Kong.

[0045] In one embodiment, the cultured influenza virus is grown and harvested from fertilized chicken eggs. Viruses can only replicate in living cells. Therefore, a cell culture system was developed for cultivating viruses in the laboratory using fertile hens' eggs. The use of eggs for virus propagation was first demonstrated by Woodruff, Goodpasture, and Burnet in 1930, and this method is well known in the art. An updated protocol can be found in "Influenza Virus Isolation and Propagation in Chicken Eggs" by Peter R. Woolcock in Method of Molecular Biology, 2008, 436:35-46. Under natural conditions, many viruses are relatively host-specific. The classical reassortant method generates fast growing, low pathogenicity viruses containing high growth properties of the A/PR/8/34 strain combined with the desired antigenic properties of a selected wild-type strain, for example, H5N1 strain. The process of generating reassortants consists of infecting embryonated chicken eggs with both strains and selecting for the virus that has the same antigenic proteins as the desired wild-type strain. Other reassortant methods using chicken eggs to produce virus are described in U. S. Patent Nos. 4,071,618, 4,318,903, 4,552757 and 4,552758, all of which are incorporated herein by reference.

[0046] Influenza A viruses contain a segmented genome that is composed of negative strand RNA, i.e., RNA having polarity opposite that of messenger RNA (mRNA) . The genome contains ten protein- coding genes, each of which has been mapped to one of the eight discrete RNA segments. Viral proteins include the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, a nucleoprotein (NP), two non-structural proteins (NSl and NS2), three polymerase polypeptides (PA, PBl and PB2), and the matrix structural proteins Ml and M2. During replication, the genome is packaged with the nucleoprotein into a helical nucleocapsid, which itself is surrounded by a lipid envelope containing the HA and NA proteins. The polymerase proteins are located inside the virion where they are complexed with the genomic RNA. As is typical of viruses with RNA genomes, the less critical regions of the viral genes are subject to genetic drift, so that sequence variation is commonly observed among virus isolates. Regions of the proteins that are critical to protein function are highly conserved.

[0047] Individual segments of the influenza virus genome can reassort when cells are co-infected with two different strains of the virus. In this fashion, genes of one strain of influenza virus can be recovered into another strain.

[0048] A typical egg culture -based protocol for preparing an influenza vaccine is set forth herein. In the infected embryonated chicken, the viruses are assembled and released into the allantoic fluid. The viruses in the allantoic fluid are harvested and inactivated with formaldehyde. The virus is then concentrated and purified in a linear sucrose density gradient solution, using a continuous flow centrifuge. The virus is then chemically disrupted using glyco p-isooctylphenyl ether (TRITON^® X-100) producing a split- antigen. The split-antigen is then further purified by chemical means and suspended in sodium phosphate -buffered isotonic sodium chloride solution. Gelatin (0.05%) is then added as a stabilizer and thimerosol (1:10,000) is added as a preservative. Other methods of recovering virus from debris- containing allantoic fluid of virus-infected chick embryos are described in U. S. Patent No. 7,270,990. [0049] In one embodiment, the cultured influenza virus comprises viruses grown and harvested from cultured eukaryotic cell lines. Mammalian cell lines include but are not limited to an African green monkey kidney cell line (VERO cells; U. S. Patent No. 5,911,998), Madin-Darby Canine Kidney (MDCK) cells (U. S. Patent Application No. 2007/0117131), CVl cells, COS-I cells, COS-7 cells, fish cell cultures (U. S. Patent No. 4,783,411) and BHK-I cells (U. S. Patent No. 5,672,485 and 5,989,805, and PCT Publication No. WO/2008/032219). Preferably the mammalian host cells are continuous cell cultures of primary, cultured epithelial cells or fibroblasts, or mammalian cell lines of passage number 10-250. Preferably, for used in vaccine production, the mammalian host cells are primary Vero cells that are a continuous cell culture of a passage number of about 20-250. These primary Vero cells are currently available and certified (e.g., by the World Health Organization, WHO). Vero cell lines are between passage number 135-190 (e.g., ATTC NO:X38).

[0050] In one embodiment, the cultured influenza virus comprises genetically engineered attenuated viruses, such as those with mutations in the modified non-coding region that down-regulates synthesis of a viral capsid gene as described in U. S. Patent No. 6,022,726. Mutations in the NP (e. g. Al 14 and H410) and one in PA (e. g. M431) confer the temperature sensitive (ts) phenotype to cold-adapted (ca) Influenza B virus strain B/Ann Arbor/1/66, whereas these same changes plus two additional residues in Ml (e. g. Q159 and V183) control the attenuated (att) phenotype. Using reverse genetics to engineer an attenuated live virus (see below), these mutations can be reassorted, for example, to the divergent wt B/Yamanashi/166/98 strain, conferring both the ts and the att phenotypes on the recombinant B/Yamanashi/166/98 (Hoffmann E, 2005, J Virol. 79:11014-21).

[0051] In one embodiment, the cultured influenza virus comprises genetically engineered attenuated viruses that provide broad cross-protection among different strains of Influenza B virus and A virus, i. e. pandemic protection. For example, Suguitan AL Jr, et. al. (2006, PLoS Med. 3(9):e360) describes a live, attenuated influenza virus possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) Nl neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/ Ann Arbor/6/60 ca (H2N2). The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. This live, attenuated influenza virus is generated by reverse genetics. Other genetically engineered attenuated influenza viruses with pandemic protection include an influenza A H5N2 reassortant described by Desheva JA, et. al. (2006, Vaccine, 24:6859-66), an attenuated H5N1 avian influenza virus vaccine generated with all eight genes from avian viruses described by Shi H., et. al, (2007, Vaccine, 25:7379-84), a live attenuated cold-adapted influenza A H7N3 virus vaccine described by Joseph T. et. al., (2008, Virology, 378:123-32). An example of a pandemic vaccine composition of the present invention comprises an inactivated Influenza virus A H5N1 strain and an isolated or recombinant influenza NP. Another example comprises an inactivated Influenza virus A H5N1 strain, an isolated or recombinant influenza NP and an isolated or recombinant influenza M2. [0052] In one embodiment, the cultured influenza virus is produced by a plasmid-based reverse genetics method. The segmented nature of the influenza virus genome allows for the traditional reassortment between two viruses in a coinfected cell. This technique has long been used to generate strains for the preparation of either inactivated or live attenuated influenza vaccines. Influenza viruses are generated entirely from cloned plasmid DNA by cotransfection of appropriate cells with 8 or 12 plasmids encoding the influenza virion sense RNA and/or mRNA. This method of producing viruses enables the routine and rapid generation of strains for either inactivated or live attenuated influenza vaccine, the generation of genetically engineered donors attenuated through directed mutation of one or more internal genes, and a means to modify genes to remove virulence determinants found in highly pathogenic avian strains. (K. Smith, 2007, MMG 445 Basic Biotechnology eJournal, 3:123-130; Subbarao and Katz, 2004, Curr. Top. Microbiol. Immunol., 283:313-42). For example, U. S. Patent No. 7,312,064 describes an approach in which a set of plasmids is introduced into a host cell, wherein each plasmid comprises one viral genomic segment, and wherein viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element for the synthesis of vRNA or cRNA with an exact 3' end. This results in expression of vRNA or cRNA, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, resulting in expression of viral mRNA and a corresponding viral protein, wherein the expression of the full set of vRNAs or cRNAs and viral proteins results in assembly of an infectious influenza virus. Multi plasmid systems for the production of influenza viruses are known in the art, see, e. g. U. S. Application Nos. 2005/0266026 and 2005/0158342.

[0053] In one embodiment, the cultured influenza virus consists essentially of inactivated influenza virus. Virus can be inactivated by suspension in chemicals such as formaldehyde (U. S. Patent Nos. 4,057,626 and 6,673,349), ethylenimine, binary ethylenimine, acetylethylenimine (U. S. Patent No. 6,803,041), hydrogen peroxide (U. S. Patent Application No. 2007/0031451) or betapropiolactone, heat- inactivation at 56°C for 30 minutes, or by high hydrostatic pressure (Luciane P. Gaspar, 2008, J. Virol. Methods, 150:57-62). Stauffer, Fausto et. al., reviewed the literature and patents related to the mechanisms used for viral inactivation, mainly chemical and physical procedures, including the strategies that are currently being explored and that have been recently described in Recent Patents on Anti- Infective Drug Discovery, 2006, l(3):291-296.

[0054] In one embodiment, the cultured influenza virus consists essentially of 'split' influenza virus. Cationic and/or non-ionic detergent can be used to open the viron particle to expose more antigenic sites of the surface antigens HA and NA (U. S. Patent No. 5,948,410). The virus is chemically disrupted using TRITON^® X-100, cetyltrimethylammoniumbromide (U. S. Patent No. 5,948,410) or sodium deoxycholate to produce a split-antigen.

[0055] In one embodiment, the cultured influenza virus consists essentially of attenuated live influenza virus. Various methods of attenuating virulent viruses are known in the art. For example, live influenza virus can be attenuated by freeze -drying (U. S. Patent No. 4,278,662). In that approach, the vaccine is prepared by allowing the virus strain to grow in the allantoic cavity of fertile hen's eggs, then harvesting and freeze-drying the resulting virus material.

[0056] Live influenza virus can also be attenuated by subjecting the virus to a large number of serial passages in a specific host tissue type at a lower temperature (See U. S. Patent Nos. 4,159,319, 4,324,861, and 7,344,722) or by mutations (A. Whiteley, et. al., 2004, International Congress Series, Titled " Options for the Control of Influenza V. Proceedings of the International Conference on Options for the Control of Influenza V",1263: 687-690; H. Caplen, et. al., 1985, J. Gen. Virol. 66:2271-2277; PCT Application No. WO/2005/012535). The prototype attenuated virus is the cold-adapted (ca) variant derived from the wild-type strain A/Ann. Arbor/6/60 (A/AA/6/60) (e.g., Cox et al., 1988, Virology 167:554-567; Hoffmann E, 2005, J. Virol., 79:11014-21, U. S. Patent No. 7,344,722). This virus is derived by serial passage of the parent strain at successively lower temperatures until a variant emerged that is capable of efficient replication at 25°C. The ca mutant has three phenotypes, namely, temperature sensitivity (ts), cold adaptation (ca), and attenuation for the respiratory tract of animals. The PBl and PB2 genes are independently responsible for the ts phenotype of the influenza A/AA/6/60 virus. Cox et al. cloned and sequenced six of the RNA segments from A/AA/6/60 and the cold-adapted variant derived from it, i.e., ca A/AA/6/60. These comparisons revealed 24 nucleotide differences between the A/AA/6/60 ca mutant and its wild-type virus parent, which resulted in amino acid changes in all six of the internal viral proteins. The PB2 protein differed from the wild-type protein in predicted amino acid sequence only at position 265, which mutation was presumed to be responsible for the ts and attenuation phenotypes specified by the A/AA/6/60 ca PB2 gene. Live vaccines have been developed that contained the HA and NA from a new epidemic variant virus and five or six of their other genes from the cold- adapted (ca) AIAAIbIbO strain.

[0057] Live attenuated influenza virus vaccines administered intranasally induce local, mucosal, cell- mediated and humoral immunity. Cold-adapted (ca) reassortment (CR) viruses containing the six internal genes of live, attenuated influenza A/ Ann Arbor/6/60 (H2N2) or B/Ann Arbor/1/66, and the hemagglutinin (HA) and neuraminidase (NA) of contemporary wild-type influenza viruses are reliably attenuated.

[0058] In addition to the PB2 mutation found in ca A/AA/6/60, other ts PB2 mutants are described in Shimizu et al.,1982, Virology, 117:38-44 and Lawson et al., 1992, Virology 191:506-510. [0059] U. S. Patent No. 6,344,354 describes a method of producing attenuated influenza virus by replicating the virus in mammalian cell culture in the presence of trypsin, at a passage number of about 20-250.

[0060] Attenuated influenza A viruses are produced by introducing coding genes of temperature sensitive or mutated viral genes into embryonated chicken eggs or cultured cell lines during the viral culture and propagation steps so that reassortment produces an attenuated virus. Examples of such strategies are described in PCT Application Nos. WO/1995/008634 and WO/2001/009291, U. S. Patent Nos. 6,669,943, 7,262,045, and 7,312,064, and U. S. Patent Application No. 2004/0109877. Mutations include but are not limited to: the replacement of the poly U tract in a genomic neuraminidase gene by a poly A tract which is capable of being copied to provide a poly U tail for mRNA transcribed from the neuraminidase gene; base pair substitution at the 5' and 3' non-coding regions adjacent to coded genes such that expression of the protein-coding sequence in cells infected by the virus is reduced to give an attenuated phenotype (see WO/1999/057284); and modifications to the NSl gene that diminish or eliminate the ability of the NSl gene product to antagonize the cellular influenza response. The mutant viruses replicate in vivo but demonstrate reduced pathogenicity (U. S. Patent Application 2004/0109877), mutations in PB2 described previously, and mutations in the two Ml residues, Q159 and V183, that are unique to Influenza B (MDV-B) and contribute to reduced virus replication at temperatures greater than 33°C, reduced Ml membrane association and its reduced virion Ml incorporation (Chen Z. et. al., 2008, Virology, 380:354-62). Such attenuated viruses are well suited for live virus vaccines and pharmaceutical formulations.

[0061] In one embodiment, the culture of influenza virus comprises a cold-adapted B/Ann Arbor/1/66 reassortant and/or a cold-adapted Arbor/6/60 (A/AA/6/60) reassortant (Chan W, et. al., 2008, Virology, 380:304-11). The cold-adapted B/Ann Arbor/1/66 strain is the master donor of the Influenza B (MDV- B) vaccine component of live attenuated influenza FLUMIST^® vaccine (Chen Z. et. al., 2008, Virology, 380:354-62). This cold-adapted live influenza virus can replicate efficiently at 25°C in the nasal passages, which are below normal body temperature. The cold-adapted B/Ann Arbor/1/66 reassortant viral strains are reassortants that are temperature sensitive (ts) so that their replication is restricted at 37°C (Type B strains) and 39°C (Type A strains) (see U. S. Patent Nos. 5,149,531 and 7,262,045; Drugs R D. 2003, 4:312-9).

[0062] Cheung TK et. al., 2005, J Gen Virol. 86: 1447-54 described the generation of recombinant Influenza A virus without M2 ion-channel protein by introduction of a point mutation at the 5' end of the viral intron. The M2 ion-channel protein is critical, but not essential, for virus replication in cell culture. This approach can provide a new way of producing attenuated Influenza A virus.

[0063] In one embodiment, the cultured influenza virus comprises a combination of Influenza A and B viruses. In one embodiment, the cultured influenza virus comprises three influenza viruses, a trivalent culture of influenza viruses. In another embodiment, the culture of influenza virus comprises two different influenza type A strains and one influenza type B strain. For example, influenza A (HlNl), A (H3N2), and B viruses. Reassortants are also considered.

[0064] In one embodiment, the cultured influenza virus comprises four influenza viruses, e. g., containing both interdemic and pandemic (H5N1) serotypes of influenza viruses. For example, Influenza virus serotypes H3N2, HlNl, B, and H5. A tetravalent culture of influenza virus is useful in the preparation of a tetra vaccine that can provide both pandemic and seasonal protection (Onishchenko GG., et. al., 2007, Zh Mikrobiol Epidemiol Immunobiol. 4:15-9).

Vaccine compositions comprising influenza antigenic polypeptides

[0065] The inventors have discovered that the combination of an isolated recombinant baculovirus- expressed influenza virus nucleoprotein (NP) polypeptide with a recombinant influenza antigenic polypeptide greatly enhanced the induced immune response and antibody production compared to the induced immune response and antibody production by the recombinant influenza antigenic polypeptide alone. In addition, the inventors found that the combination of an isolated recombinant baculovirus- expressed influenza virus nucleoprotein (NP) and matrix (M) proteins with a recombinant influenza antigenic polypeptide significantly enhanced the induced immune response and antibody production. The M protein can be an influenza virus Ml or M2 protein. Accordingly, isolated recombinantly expressed or isolated influenza virus nucleoprotein (NP) and matrix (M) proteins can be useful in vaccine preparations for immunization protection against influenza infections.

[0066] Accordingly, embodiments of the invention provide vaccine compositions that have improved immungencity, comprising an influenza antigenic polypeptide and an isolated influenza virus nucleoprotein (NP) polypeptide.

[0067] Additionally, embodiments of the invention provide vaccine compositions that have improved immungencity, comprising an influenza antigenic protein, an isolated influenza virus matrix (M) protein and an isolated influenza virus nucleoprotein (NP) polypeptide. [0068] In one embodiment, the isolated influenza virus matrix (M) protein is an Ml protein.

[0069] In another embodiment, the isolated influenza virus matrix (M) protein is an M2 protein.

[0070] In yet another embodiment, the isolated influenza virus matrix (M) protein is an M2e protein. In additional embodiments, various combinations of isolated M proteins can be included in the vaccine compositions.

[0071] Accordingly, in one embodiment, provided are vaccine compositions that have improved immungencity, comprising an influenza antigenic polypeptide, an isolated influenza virus Ml protein and an isolated influenza virus nucleoprotein (NP) polypeptide.

[0072] In another embodiment, provided are vaccine compositions that have improved immungencity, comprising an influenza antigenic polypeptide, an isolated, influenza virus M2 protein and an isolated, influenza virus nucleoprotein (NP) polypeptide.

[0073] In another embodiment, provided are vaccine compositions that have improved immungencity, comprising an influenza antigenic polypeptide, an isolated influenza virus Ml protein, an isolated influenza virus M2 protein, and an isolated influenza virus nucleoprotein (NP) polypeptide.

[0074] In another embodiment, provided are vaccine compositions that have improved immungencity, comprising an influenza antigenic polypeptide, an isolated influenza virus Ml protein, an isolated influenza virus M2e protein, and an isolated influenza virus nucleoprotein (NP) polypeptide, that can be useful in vaccine preparations for immunization against influenza infections.

[0075] In some embodiments, the isolated influenza virus Ml, M2, M2e and NP are fusion polypeptides. In a preferred embodiment, the isolated influenza virus Ml, M2, M2e and NP are Bacillus anthracis Lethal Factor (LF) fusion polypeptides, i. e. influenza virus Ml, M2, M2e or NP fused to the B. anthracis LF protein. In another preferred embodiment, the isolated influenza virus Ml, M2, M2e and

NP are B. anthracis N-terminal Lethal Factor (LFn) fusion polypeptides, i. e. influenza virus Ml, M2,

M2e or NP fused to the B. anthracis LFn protein. The influenza virus Ml, M2, M2e and NP can also be fused to fragments of the B. anthracis LF protein, fragments that are at least 15 amino acids residues in length. The fusion is via a peptide bond between the viral protein and the LF protein or fragment.

[0076] In one embodiment, the immungencity induced includes both innate and acquired immunity.

[0077] In one embodiment, the influenza antigenic polypeptide is a polypeptide isolated, extracted and/or purified from an influenza virus. The art of fractionation of influenza virus is well known, e. g. as described in WO/ 1998/004242; Pease LF 3^rd, et. al., 2009, Biotechnol Bioeng. 102:845-55; Glushakova

SE et al., 1988, Vopr. Virusol., 33:286-9; Bottex C. et al. 1975, Arch. Virol. 48:9-19; Hoyle L. et al.

1973, Postgrad. Med. J., 49:193-4; and Taikova NV. et al. 1971, Mikrobiol Zh., 33:334-8. Detergent and/or enyzymes can be used to expose additional antigenic and/or immunogenic sites on the influenza polypeptide, e. g. ammonium deoxycholate and carboxypolypeptidase treatment. In one embodiment, the influenza virus is Influenza A virus or Influenza B virus.

[0078] In one embodiment, the virus from which the influenza antigenic polypeptide is derived from is a wild type isolate, e. g. A/Ck/Hidalgo/26654- 1368/94 (Mex. Isolate), A/Hong Kong/156/97 (H5N1) and

Influenza B virus B/Yamagata. In one embodiment, the virus from which the influenza antigenic polypeptide is derived from is a reassortant influenza virus. In another embodiment, the virus from which the influenza antigenic polypeptide is derived is a genetically engineered reassortant influenza virus. Such a virus can have genetic mutations in the coding sequences of any of the ten viral proteins, e. g. in NSl, PB2, and M2 proteins described previously.

[0079] In one embodiment, the influenza antigenic polypeptide is a recombinant polypeptide, expressed by molecular methods. Molecular methods for making recombinant proteins are well known in the art. Such methods include but are not limited to polymerase chain reaction (PCR) cloning of the gene for the desired influenza antigenic polypeptide, construction of an expression plasmid for the selected influenza antigenic polypeptide, transfection of host cells with the expression plasmid, and induced protein expression in the transformed host cells. In one embodiment, the expression plasmid for the selected influenza antigenic polypeptide allows expression of the selected influenza antigenic polypeptide in a variety of host cells, e. g. bacteria, yeast, insect, fish, and mammal. Recombinant HA of A/Texas/36/91, expressed in a baculovirus expression vector system (BEVS), is reported in U. S. Patent Nos. 5,858,368 and 7,399,840. Recombinant M2 of A/ Ann Arbor/6/60, expressed in a baculovirus expression vector system (BEVS), is reported in U. S. Patent No. 5,290,686. Recombinant neuraminidase (rNA) is reported in U. S. Patent No. 5,976,552.

[0080] In one embodiment, the recombinant influenza antigenic polypeptide is expressed and purified from bacteria. In another embodiment, the recombinant influenza antigenic polypeptide is expressed and purified from a baculovirus expression vector system (BEVS), e. g. in Lepidopteran cells: SF9, SF21, High-5, Mimic-SF9; or from a Drosophila Stable Expression System (DES) using S2 Schneider cells or D.Mel2 cells. For example, in U. S. Patent No. 5,762,939, a recombinant hemagglutinin polypeptide that is full-length, uncleaved (HAO), glycoprotein is produced from baculovirus expression vectors in cultured insect cells and purified under non-denaturing conditions.

[0081] In one embodiment, the influenza antigenic polypeptide is a fragment, a peptide of at least 6 amino acids, mutant and/or variant thereof of the gene encoded full-length polypeptide. For example, the full-length M2 protein as encoded by the M2 gene consists of an extracellular domain (amino acid residues 1-23), a transmembrane domain (amino acid residues 24-44), and a cytoplasmic domain (amino acid residues 45-97). The DNA sequences of the M2 genes of numerous influenza A viruses are known (Ito et. al. 1991, J. Virol. 65:5491-5498). Using molecular methods, a fragment of M2 can be made, consisting of the extracellular domain (amino acid residue 1-24), commonly termed as M2e. De Filette M. et. al., (2006, Vaccine, 24:6597-601) describes an M2e-based human Influenza A vaccine. Another fragment of M2 can be the cytoplasmic domain (amino acid residues 45-97). Yet another fragment of M2 can consist of the extracellular domain (amino acid residues 1-24) and the cytoplasmic domain (amino acid residues 45-97), with the transmembrane domain deleted. This fragment of M2 is a soluble protein. WO99/28478 describes a vaccine containing a soluble M2. The transmembrane domain (amino acid residues 26-43) is deleted. Additional amino acids flanking the transmembrane domain can be removed too. WO99/28478 also describes a vaccine containing a soluble M2 where the transmembrane domain and additional flanking C-terminal region (amino acid residue 26-55) is deleted. WO99/28478 also describes a vaccine containing a soluble M2 where the deleted transmembrane domain is replaced with glycine amino acid residues. U. S. Patent Application 2008/0008725 describes soluble hemagglutinin (HA) comprising the N-terminal globular head, termed HA ectodomains. These soluble HA are missing the secretion signal peptide, amino acid residues 1-16, and are C-terminally truncated at Arg 329 for H3 and Arg 326 for H5. C-terminal truncation eliminates the C-terminal anchor in HA. In U. S. Patent Nos. 5,762,939 and 5,858,368, the furin cleavage site of the full-length HA is deleted to prevent cleavage when expressed in insect cells. WO 2007/100584 describes two recombinant truncated HA polypeptides: a "short HA" has the C-terminal anchor and additional 10 amino acids upstream eliminated; and the "long HA" has just the C-terminal anchor eliminated. Both "short HA" and "long HA" have modifications at the furin cleavage site, where the first five basic amino acids are deleted. [0082] In one embodiment, the influenza antigenic polypeptide, fragment, peptide of at least 6 amino acids, mutant and/or variant thereof can have changes in the amino acid sequences, e. g. conservative amino acid substitution or non-conservative amino acid substitution. Such amino acid sequence changes can improve protein expression in the host cells /expression system. In WO99/28478, the M2 proteins and fragments have substitution of hydrophilic or neutral amino acids for hydrophobic amino acids to enhance expression in the prokaryotic and/or eukaryotic system and also to render the mutant M2 more soluble in aqueous solution relative to the native M2.

[0083] Amino acid substitution can also be done for the purpose of eliminating specific recognition sites. In U. S. Patent Application 2008/0008725, the furin cleavage site of the full-length HA, RKKR (SEQ. ID. No. 18), is mutated to KNTR (SEQ. ID. No. 32), to prevent cleavage when expressed in S2 cells.

[0084] In one embodiment, the influenza antigenic polypeptide, fragment, peptide of at least 6 amino acids, mutant and/or variant thereof can have additional amino acid sequences. U. S. Patent Nos. 5,762,939 and 5,858,368 describe a matured recombinant full-length hemagglutinin (HAO) protein expressed from a baculovirus expression vector system. The N-terminal signal peptide is replaced with a chitin signal peptide, a baculovirus signal peptide, to facilitate entry into the endoplasmic reticulum of the insect cells.

[0085] The mature HA forms homotrimers. In WO 2007/100584, recombinant HA has oligomerization motifs (termed a foldon) inserted in the polypeptide. Additionally, the recombinant HA-foldon polypeptide has a histidine tag for protein purification purposes. Several exogenous oligomerization motifs have been successfully used to promote stable trimers of soluble recombinant proteins: the GCN4 leucine zipper (Harbury et al. 1993, Science 262:1401-1407), the trimerization motif from the lung surfactant protein (Hoppe et al. 1994, FEBS Lett. 344:191-195), collagen (McAlinden et al., 2003, J. Biol. Chem. 278:42200-42207), and the phage T4 fibritin 'foldon' (Miroshnikov et al., 1998, Protein Eng., 11:329-414). The fibritin foldon, a 27 amino acid sequence

(GYIPEAPRDGQAYVRKDGEWVLLSTF, SEQ. ID. NO. 24), adopts a β-propeller conformation, and can fold and trimerize in an autonomous way (Tao et al. 1997, Structure 5:789-798). It has been reported recently that this foldon can successfully induce stable trimerization of other fibrous motifs such as phage

T4 short-tail fibers and adenovirus fibers, as well as viral human immunodeficiency virus glycoprotein gp 140.

[0086] In one embodiment, the influenza antigenic polypeptide is a peptide of at least 6 amino acids, mutant and/or variant thereof. The peptide comprises the epitope displayed on a MHC class I molecule or a class II molecule when the full-length influenza antigenic polypeptide is processed by a MHC class I or

II presenting cell. Examples of such epitopes are: the influenza virus HA MHC class II epitope (aa 91-

108); HA MHC class I epitope (aa307-319); and NP MHC class I NP (aa335-350) epitope and the NP

(aa380-393). Additional epitope for Ml, M2, HA, NP, and PB peptide epitopes can be found in U. S.

Patent No. 6,740,325, WO 2007/082734, PCT/US2008/067001 and WO 2007/066334.

[0087] In one embodiment, the peptide is at least 6 amino acid residues and up to 15 amino acid residues.

[0088] In one embodiment, the peptide epitopes of an influenza antigenic polypeptide is fused to another polypeptide. In U. S. Patent No. 6,740,325, the peptide epitopes of HA and NP are fused to a

Salmonella flagellin.

[0089] In one embodiment, the influenza antigenic polypeptide is a recombinant polypeptide comprising multiple peptide epitopes fused together. Fusion of multiple peptide epitopes forms a polytope. U. S. Patent No. 6,740,325 and WO 2007/082734 describe chimeric polytope influenza antigenic polypeptides, e. g. HA (aa 91-108)-HA (aa307-319)-NP (aa335-350)-NP (aa380-393)- flagellin fusion polypeptide.

[0090] In one embodiment, the chimeric polytope comprises 2-10 peptide epitopes, 5-20 peptide epitopes, or 10-40 peptide epitopes. In some embodiments, the peptide epitopes are derived from any of the ten influenza polypeptides. In some embodiments, the peptide epitopes are derived from any of the

Influenza A virus subtypes, e. g. HlNl, H5N1, H3N2, H7N3 etc. The peptide epitopes compiled in the polytope can be according to the Influenza A and B viruses subtypes predicted for the upcoming flu season.

[0091] In one embodiment, the influenza antigenic polypeptide is a chimeric polypeptide, comprising different influenza antigenic polypeptides, for example, M2 fused with NP. Heinen PP. et. al., (2002, J

Gen Virol. 83:1851-9) describes a vaccine containing M2e fused to the influenza A virus nucleoprotein

(M2eNP). The chimeric polypeptide can comprise full-length influenza protein components. Conversely, the chimeric polypeptide can comprise fragments of the influenza protein components. Preferably, the fragments are known antigenic fragments, e. g. the ectodomain of NA, HA and M2e. Additionally, the chimeric polypeptide can comprise antigenic epitopes of the influenza protein components, e. g. HA

(306-322) epitope and NA (190- 230). US Patent No. 5,858,368 describes a recombinant HA-NA fusion polypeptide. Other chimeric polypeptides contemplated include but are not limited to HA-Ml, HA-M2,

NA-Ml, NA-M2, NA-HA-M2e etc.

[0092] In one embodiment, the influenza antigenic polypeptide is fused to a non-influenza polypeptide.

For example, De Filette M. et. al., describes vaccines containing M2e -fusion proteins (Vaccine, 2006, 24:6597-601; Virology, 2005, 337:149-61; Heinen PP. et. al., 2002, J Gen Virol. 83:1851-9). M2e was fused to hepatitis B virus core protein (M2eHBc). This M2e-HBc vaccine induced complete protection in mice against a lethal influenza challenge. Protective immunity was obtained regardless of the position of M2e in the M2e-HBc chimera at the amino-terminus or inserted in the immuno-dominant loop of the HBc protein. Examples of non-influenza polypeptide include but are not limited to bacterial toxins and subunits thereof, viral proteins or subunits thereof (e. g. hepatitis B viral protein, HIV protein), bacterial structural proteins, e. g. flagellin, and a carcinoembryonic antigen. In WO 2006/123155 and WO 2007/062832, the influenza antigenic polypeptides Ml, M2, NP, NSl, NS2, PBl, PB2, PA, HA and NA are fused to the B-subunit of Escherchia coli heat labile toxin. Other toxins include the amino terminal domain of the anthrax lethal factor (LF), Pseudonomas aeruginosa exotoxin A, and the adeylate cyclsae A from B. pertussin. Fusion with the toxin subunits improved MHC I class presentation of the antigen. In U. S. Patent No. 6,740,325, peptide epitopes of HA and NP are fused to a Salmonella flagellin. Vaccines comprising fusion proteins linking flagellin with the M2e or the globular head domain of HA have been described and are in clinical trials conducted by Vaxlnnate.

[0093] The amino-terminal domain from B. anthracis (anthrax) LF is known as LFn. It is the N- terminal 255 amino acids of LF. LF has been found to contain the information necessary for binding to protective antigen (PA) and mediating translocation. The domain alone lacks lethal potential, which depends on the putatively enzymatic carboxyl-terminal moiety (Arora and Leppla 1993, J. Biol. Chem., 268:3334-3341).

[0094] In one embodiment, the influenza antigenic polypeptide is conjugated to a carrier molecule which potentiates an immune response to the HA protein. The cross-link can be, e.g., via free sulfhydryl group in the endodomain of the isolated whole HA protein. Methods of forming such conjugates are described in U. S. Patent No. 5,612,037.

[0095] It is also contemplated that the vaccine composition described herein can comprise a plurality of any of the influenza antigenic polypeptides described herein. Preferably, the vaccine composition comprises at least three influenza antigenic polypeptides, e.g. a recombinant HA, a recombinant NA, and a fusion chimeric polytope polypeptide. [0096] Non-limiting vaccine compositions comprising inactivated or attenuated cultured virus are:

1. inactivated Influenza A (HlNl), inactivated Influenza A (H3N2), inactivated Influenza B/Florida, and full-length baculo virus-expressed Influenza A NP (SEQ. ID No.6);

2. inactivated Influenza A (HlNl), inactivated Influenza A (H7N7), inactivated Influenza B/Florida, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6) and full-length baculo virus-expressed Influenza A MI (SEQ. ID. No. 7);

3. inactivated Influenza A (H3N2), inactivated Influenza A (H7N7), inactivated Influenza B/Victoria, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6), full-length baculovirus-expressed Influenza A MI (SEQ. ID. No. 7) and full-length baculovirus-expressed Influenza A M2 (SEQ. ID. No. 8); 4. inactivated Influenza A (H3N2), inactivated Influenza A (H7N3), inactivated Influenza B/Victoria, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6), full-length baculo virus-expressed Influenza A MI (SEQ. ID. No. 7) and baculovirus-expressed Influenza A M2e (amino acid 1-23 of SEQ. ID. No. 8);

5. inactivated Influenza A (H3N2), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6) and baculovirus-expressed Influenza A M2e (amino acid 1-23 of SEQ. ID. No. 8);

6. inactivated Influenza A (H3N2), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, and baculovirus-expressed Influenza A LFn-NP fusion protein;

7. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus- expressed Influenza A M2e (amino acid 1-23 of SEQ. ID. No. 8);

8. inactivated Influenza A (H5N3), inactivated Influenza A (H7N2), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus- expressed Influenza A Ml (SEQ. ID. No. 7);

9. inactivated Influenza A (H5N1), inactivated Influenza A (H7N2), inactivated Influenza B/Yamagata, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus- expressed Influenza A M2 (SEQ. ID. No. 8);

10. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus- expressed Influenza A LFn-M2e-Ml fusion protein; and

11. inactivated Influenza A (H3N2), inactivated Influenza A (H7N7), inactivated Influenza B/Shanghai, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus- expressed Influenza A LFn-M2e fusion protein.

[0097] Examplary pandemic vaccine compositions comprising inactivated or attenuated cultured virus are:

1. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, and baculovirus-expressed Influenza A LFn-NP fusion protein;

2. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A LFn-NP fusion protein; and baculovirus- expressed Influenza A LFn-M2 fusion protein;

3. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, and baculovirus-expressed Influenza A NP protein;

4. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A NP protein and baculovirus-expressed Influenza A M2 protein; 5. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A NP protein; and baculovirus-expressed B. anthracis LFn protein; and

6. inactivated Influenza A (H5N1), inactivated Influenza A (H7N3), inactivated Influenza B/Hongkong, baculovirus-expressed Influenza A NP protein; baculovirus-expressed Influenza A M2 protein and baculovirus-expressed B. anthracis LFn protein

[0098] Non-limiting vaccine compositions comprising influenza antigenic polypeptides are:

1. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, and full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6);

2. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6) and full-length baculovirus-expressed Influenza A Ml (SEQ. ID. No. 7);

3. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6) and full-length baculovirus-expressed Influenza A M2 (SEQ. ID. No. 8);

4. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6), full-length baculovirus-expressed Influenza A Ml (SEQ. ID. No. 7) and full- length baculovirus-expressed Influenza A M2 (SEQ. ID. No. 8);

5. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6), full-length baculovirus-expressed Influenza A Ml (SEQ. ID. No. 7) and baculovirus-expressed Influenza A M2e (amino acid 1-23 of SEQ. ID. No. 8);

6. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, full-length baculovirus-expressed Influenza A NP (SEQ. ID No.6) and baculovirus-expressed Influenza A M2e (amino acid 1-23 of SEQ. ID. No.

8);

7. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, fusion proteins HA-M2 and NA-Ml of Influenza A subtypes and baculovirus-expressed Influenza A LFn-NP fusion protein;

8. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, and baculovirus-expressed Influenza A LFn-NP fusion protein;

9. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, baculovirus-expressed Influenza A LFn-NP fusion protein, and baculovirus-expressed Influenza A LFn-M2 (SEQ. ID. No. 8); 10. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus-expressed Influenza A M2e (amino acid 1-23 of SEQ. ID. No. 8);

11. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus-expressed Influenza A MI (SEQ. ID. No. 7);

12. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus-expressed Influenza A M2 (SEQ. ID. No. 8);

13. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus-expressed Influenza A LFn-M2e-Ml fusion protein;

14. Influenza A (H3N2) full-length HA and NA, Influenza A (H7N7) full-length HA and NA, Influenza B/Shanghai full-length HA and NA, baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus-expressed Influenza A LFn-M2e fusion protein;

15. Influenza A (H3N2) HA (aa 91-108)-HA( aa307-319)-NP (aa335-350)-NP (aa380-393)-flagellin fusion polypeptide, Influenza A (H7N7) fusion protein HA-M2 and NA-M2, Influenza B/Shanghai flagellin fusion HA and flagellin fusion NA, and baculovirus-expressed Influenza A LFn-NP fusion protein and baculovirus-expressed Influenza A LFn-M2e fusion protein;

16. Influenza A (H3N2) full-length HA, Influenza A (H7N7) full-length HA, Influenza B/Shanghai full-length HA, and baculovirus-expressed Influenza A NP (SEQ. ID. No. 6);

17. Influenza A (H3N2) full-length HA, Influenza A (H7N7) full-length HA, Influenza B/Shanghai full-length HA, baculovirus-expressed Influenza A NP (SEQ. ID. No. 6) and baculovirus- expressed Influenza A M2 (SEQ. ID. No. 8);

18. Influenza A (H3N2) full-length HA, Influenza A (H7N7) full-length HA, Influenza B/Shanghai full-length HA, and baculovirus-expressed Influenza A LFn-NP;

19. Influenza A (H3N2) full-length HA, Influenza A (H7N7) full-length HA, Influenza B/Shanghai full-length HA, baculovirus-expressed Influenza A LFn-NP and baculovirus-expressed Influenza A LFn-M2;

20. Influenza A (H5N1) full-length NA, Influenza A (H7N7) full-length NA, Influenza B/Shanghai full-length NA, and baculovirus-expressed Influenza A NP (SEQ. ID. No. 6);

21. Influenza A (H5N1) full-length NA, Influenza A (H7N7) full-length NA, Influenza B/Shanghai full-length NA, baculovirus-expressed Influenza A NP (SEQ. ID. No. 6) and baculovirus- expressed Influenza A M2 (SEQ. ID. No. 8);

22. Influenza A (H5N1) full-length NA, Influenza A (H7N7) full-length NA, Influenza B/Shanghai full-length HA, and baculovirus-expressed Influenza A LFn-NP; and 23. Influenza A (H5N1) full-length NA, Influenza A (H7N7) full-length NA, Influenza B/Shanghai full-length NA, baculovirus-expressed Influenza A LFn-NP and baculovirus-expressed Influenza A LFn-M2.

Isolated influenza virus nucleoprotein (NP) polypeptide and matrix (M) proteins

[0100] In one embodiment, the vaccine composition comprises a mixture of isolated influenza NP and Ml polypeptides. In another embodiment, the vaccine composition comprises a mixture of isolated influenza NP and M2 polypeptides. In yet another embodiment, the vaccine composition comprises a mixture of isolated influenza NP, Ml and M2 polypeptides.

[0101] Also contemplated are vaccine compositions comprising a combination of isolated influenza NP, Ml and M2 polypeptides, conservative substitution mutants thereof or fragments of at least 6 amino acids thereof, wherein at least one NP polypeptide is included.

[0102] In another embodiment, the vaccine composition comprises a pharmaceutically acceptable carrier, a B. anthracis Lethal Factor (LF) polypeptide and an influenza virus nucleoprotein (NP) polypeptide. In an additional embodiment, the vaccine composition further comprises an influenza virus matrix protein (M). The M protein can be an influenza virus Ml or M2 protein. In some embodiments, the vaccine composition comprises a mixture of individual LF, NP and Ml polypeptides, or a mixture of individual LF, NP and M2 polypeptides. In one embodiment, the vaccine composition comprises a mixture of individual LF, NP, Ml and M2 polypeptides.

[0103] In a further embodiment, the vaccine composition comprises an LF polypeptide fused with an influenza NP and/or LF polypeptide fused with an influenza M protein. In other words, the vaccine composition can comprise LF-NP and/or LF-Ml, LF-M2e and/or LF-M2 fusion polypeptides. The LF polypeptide can be the N-terminal LF polypeptide (LFn). In one embodiment, the LFn polypeptide of any vaccine composition described herein is fused to an influenza virus NP polypeptide, a conservative substitution mutant thereof or a fragment thereof of at least 6 amino acids. In another embodiment, the LFn polypeptide of any vaccine composition described herein is fused to an influenza virus M polypeptide, a conservative substitution mutant thereof or a fragment thereof of at least 6 amino acids. The M protein can be an influenza virus Ml or M2 protein. The fusion polypeptides can be formed by chemical cross-linking of individual LF polypeptide with individual NP, Ml or M2 proteins, methods for which are well known in the art (e. g. as described by Das and Fox in Annual Review of Biophysics and Bioengineering, 1979, 8:165-193). Preferably, the fusion polypeptides are synthesized using recombinant molecular methods, from single coding DNA sequences and the fusion between the LF and the influenza proteins is by way of a covalent peptide bond. Methods of making fusion proteins using recombinant molecular methods are known. One of ordinary skill in the art can clone and ligate the necessary coding DNA sequences and express fusion proteins.

[0104] Also contemplated are vaccine compositions comprising a combination of individual LF, NP, Ml and M2 polypeptides, LF-NP, LF-Ml and LF-M2 fusion polypeptides, conservative substitution mutants thereof or fragments of at least 6 amino acids thereof, wherein at least one NP polypeptide and one LF polypeptide are included.

[0105] In some embodiments, the vaccine compositions described herein do not include any nucleic acids encoding the NP or M proteins.

[0106] Typically when designing a protein vaccine against a pathogen, an extracellular protein or one exposed to the environment on a virus is often the ideal candidate as the antigen component in the vaccine. Antibodies generated against that extracellular protein become the first line of defense against the pathogen during infection. The antibodies bind to the protein on the pathogen to facilitate antibody opsonization and marks the pathogen for ingestion and destruction by a phagocyte such as a macrophage. Antibody opsonization can also kill the pathogen by antibody-dependent cellular cytotoxicity. The antibody triggers a release of lysis products from cells such as monocytes, neutrophils, eosinophils, and natural killer cells. For influenza vaccine, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are generally the antigens of choice. Both nucleoprotein (NP) polypeptide and matrix (M) are internal viral proteins and therefore not usually considered in vaccine design for antibody- based immunity.

[0107] Several studies have identified the NP antigen as a major target of the cross-reactive cytotoxic T-cell (CTL) response against influenza viruses instead of antibody-based response (Townsend et al., 1984, J. Exp. Med. 160:552-583). CTLs recognize and kill infected cells expressing viral antigens, including fragments of proteins such as NP. Therefore, cellular immunity to NP can contribute to recovery from influenza infection (Wraith et al., 1987, J. Gen. Virol.. 68:433-440). Bettosini et. al. ( J.Virol., 2005, 79:15537-15546) showed that the C terminus of the NP of Influenza A virus can be delivered as an antigen to the trans-Golgi Network and promotes an efficient presentation through MHC Class I, a major component for CTL response. Researchers at Merck and Vical Inc. reported that a vaccine based on NP partially protected mice from seasonal Influenza A, although some animals still died. Instead of immunizing the animals with NP itself, the researchers used DNA encoding the protein as the vaccine, a strategy that often generates a more powerful cellular immune response. However, immunization with vaccines containing DNA encoding NP alone did not protect mice against H5N2 influenza virus (WO 2007/016598). Altstein, et. al., 2006, reported that a two-fold immunization of Balb/c mice with a Vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (HlNl) virus under the control of a strong synthetic promoter did not induce specific antibodies or protect the animal. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection (Altstein, et. al., 2006, Archives of Virology, 151: 921-931). [0108] In one embodiment, the vaccine compositions described herein, when administered to mice, provoke an immune response that prevents death in at least 20% of animals challenged with 5 LD₅₀ of an H5N2 influenza virus or an influenza virus having the same serotype as the source of the virus-derived components in the vaccine composition, e .g. the serotype of the influenza virus from which the HA polypeptide was isolated or the serotype of the inactivated/attenuated influenza virus. Methods of vaccination and challenging an immunized animal with viral infection are known to one skilled in the art. For example, a 10 μg aliquot of inactivated/attenuated virus and/or isolated influenza antigenic polypeptide vaccine compositions comprising isolated influenza NP and/or M protein is prepared in 100 μl PBS or in incomplete Freund's adjuvant and injected intramuscularly per mouse per vaccination. Alternatively, parenteral, intraperitoneal and footpad injections can be used. Volumes of footpad injections are reduced to 50 μl. The mice are immunized with the vaccine compositions on three separate occasion with 14 days interval in between. Seven days after, the immunized mice are challenged intranasally with virus. Ether anaesthetized mice (10-12 g) are infected intranasally with 50 μl of PBS- diluted allantoic fluid containing 5 LD₅₀ of influenza A/Mallard/Pennsylvania/10218/84 H5N2 virus. Protection is measured by monitoring animal survival and body weight, which is assessed throughout an observation period of 21 days. Severely affected mice are euthanized. One LD₅₀ of A/Mallard/Pennsylvania/10218/84 is equal to 100-1000 the Tissue Culture Infectious Dose50 (TCID50) assay. In other embodiments, other stains of influenza viruses can be used to challenge the immunized mice, e . g. A/PR/8/34 (HlNl). One skilled in the art would be able to determine the LD₅₀ and TCID50 of the strain used by methods known in the art, for example as described by LaBarre and Lowy (2001) (J. Virol. Methods 96:107-26) and by Orville J. Golub (1948) (J. Immunol.,59:71-82) which are incorporated hereby reference in their entirety.

[0109] In one embodiment, the vaccine compositions described herein have an antigen that consists of the influenza virus NP polypeptide of SEQ ID NO: 6, a conservative substitution mutant thereof or a fragment of at least 6 amino acids thereof. The three species of influenza viruses all have NP, Ml and M2 proteins, and these proteins can have differences between species and differences between strains and isolates within a species. The amino acid sequence homology within a species, i. e. among the strains or virus subtype or isolates within a species is much greater than between species. For example, the amino acid sequence homology among NPs from influenza A strains is about 90%, whereas the amino acid sequence homology between the NPs from influenza A and B is about 35% over the aligned regions (Stevens and Barclay, J. Virol, 1998, 72:5307-5312). The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues.

[0110] The influenza NP useful in the methods and compositions described herein is a protein having more than 90% amino acid sequence homology to an influenza NP protein. In one embodiment, the influenza NP is 90% amino acid sequence homology to SEQ. ID. No. 6. Preferably, the full or partial region of the NP used for the present invention are as described in SEQ. ID. No. 6. Where the partial region of influenza NP is used, the amino acid sequence includes more than the N-terminal 50% or more than the C- terminal 50% of influenza NP protein. Fragments of NP of at least 6 amino acids include but are not limited to RFYIQMCTELKLSDYEGRLIQNSL (SEQ. ID. NO. 9) ,

AAFEDLRLLSFIRGTKVSPRGKLST (SEQ. ID. NO. 10), and GSSTLELRSGYWAIRTRSGGNTN (SEQ. ID. NO. 11). Methods for vaccine epitope selection in a protein are known in the art, for example, as described in references: Bastin J. et al., 1987, J. Exp. Med. 165:1508; Cossins, J. et al., 1993, Virol, 195:851; and Macken, C. et al. 2001, in "The value of a database in surveillance and vaccine selection." in Options for the Control of Influenza IV. A.D.M.E. Osterhaus, N. Cox & A.W. Hampson (Eds.) Amsterdam: Elsevier Sci. These references are incorporated herein by reference in their entirety. [0111] In one embodiment, the NP does not include the amino acids 400-498 of the full-length NP polypeptide.

[0112] In one embodiment, the vaccine compositions described herein have an antigen that consists of the influenza virus Ml polypeptide of SEQ. ID No. 7, a conservative substitution mutant thereof or a fragment of at least 6 amino acids thereof. The influenza Ml in the compositions and methods described herein is a protein having at least 90% amino acid sequence homology to an influenza Ml protein. In one embodiment, the influenza Ml has at least 90% amino acid sequence homology to SEQ. ID. No. 7. Preferably, the full or partial region of Ml used for the methods and compositions described herein are as described in SEQ. ID. No. 7. Where a partial region of influenza Ml is used, the amino acid sequence preferably includes more than the N-terminal 50% or more than the C- terminal 50% of influenza Ml protein. Fragments of Ml of at least 6 amino acids include but are not limited to SIVPSGPLKAEIAQRLEDVFAGK (SEQ. ID. NO. 12) and SPLTKGILGFVFTLTVPSER (SEQ. ID. No. 13).

[0113] In one embodiment of the vaccine compositions described herein, the antigen consists of the influenza virus M2 polypeptide of SEQ ID NO: 8, a conservative substitution mutant thereof or a fragment of at least 6 amino acids thereof. The influenza M2 in the compositions and methods described herein is a protein having at least 90% amino acid sequence homology to an influenza M2 protein. In one embodiment, the influenza M2 is 90% amino acid sequence homology to SEQ. ID. No. 8. Preferably, the full or partial region of M2 used for the compositions and methods described herein are as described in SEQ. ID. No. 8. Where a partial region of influenza M2 is used, the amino acid sequence preferably includes more than the N-terminal 50% or more than the C- terminal 50% of influenza M2 protein. Fragments of NP of at least 6 amino acids include but are not limited to

MSLLTEVETPIRNEWECRCNGSSD (SEQ. ID. NO. 14); MSLLTEVETPIRNBWGCRCNDSSD (SEQ. ID. NO. 15); and MSLLTEVETPIRNEWGCRCNGSSD (SEQ. ID NO. 16).

[0114] In one embodiment of the invention described herein, the influenza NP, the Ml protein and the M2 proteins are all wild type proteins, as in the sequences found in naturally occurring viruses and have not been altered by selective growth conditions or molecular biological methods. [0115] In one embodiment, the vaccine compositions described herein comprise an LF polypeptide. The LF polypeptide can be an N-terminal (LFn) polypeptide, or conservative substitution variant thereof, that promotes transmembrane delivery to the cytosol of an intact cell. The amino-terminal domain from B. anthracis LF polypeptide is known as LFn. LF binds to protective antigen (PA) and mediates translocation across the cell membrane. The LFn alone lacks lethal potential, which depends on the putatively enzymatic carboxyl-terminal moiety (Arora and Leppla, 1993, J. Biol. Chem., 268:3334- 3341). While not wishing to be bound by theory, the LF polypeptide, individually or fused, is thought to function to mediate membrane translocation. It has been shown that a fusion protein of the LFn domain with a foreign antigen can induce CD8 T cell immune responses even in the absence of PA (Kushner, et. al. 2003, PNAS, 100:6652-6657). The LFn polypeptide is a polypeptide comprising the amino acid residues 1-288 of the LF polypeptide and is capable of traversing cell membranes in the absence of the B. anthracis protective antigen (PA). Amino acids 1-288 includes the N-terminal leader sequence. In addition, when a second protein is attached to an LFn or LF polypeptide, this second protein is also transported across membranes into the cytosol along with the LFn or LF polypeptide. Thus, LFn can be used without PA as a carrier to deliver antigens into the cytosol. The LFn or LF polypeptide therefore facilitates and promotes the transmembrane delivery of other proteins.

[0116] In one embodiment, the vaccine compositions described herein comprise glycosylated proteins. In other words, the LF, NP, and M proteins can each be glycosylated proteins. In one embodiment of the vaccine compositions described herein, individual or fusion polypeptides are O-linked glycosylated. In another embodiment of the vaccine compositions described herein, individual or fusion polypeptides are N-linked glycosylated. In yet another embodiment of the vaccine compositions described herein, individual or fusion polypeptides are both O-linked and N-linked glycosylated. In other embodiments, other types of glycosylations are possible, e. g. C-mannosylation. In one embodiment of the vaccine compositions described herein, the LFn polypeptide is N-glycosylated. Glycosylation of proteins occurs predominantly in eukaryotic cells. N-glycosylation is important for the folding of some eukaryotic proteins, providing a co-translational and post-translational modification mechanism that modulates the structure and function of membrane and secreted proteins. Glycosylation is the enzymatic process that links saccharides to produce glycans, and attaches them to proteins and lipids. In N-glycosylation, glycans are attached to the amide nitrogen of asparagine side chain during protein translation. The three major saccharides forming glycans are glucose, mannose, and N-acetylglucosamine molecules. The N- glycosylation consensus is Asn-Xaa-Ser/Thr, where Xaa can be any of the known amino acids. O-linked glycosylation occurs at a later stage during protein processing, probably in the Golgi apparatus. In O- linked glycosylation, N-acetyl-galactosamine, O-fucose, O-glucose, and/or N-acetylglucosamine is added to serine or threonine residues. One skilled in the art can use bioinformatics software such as NetNGlyc 1.0 and NetOGlyc Prediction softwares from the Technical University of Denmark to find the N- and O- glycosylation sites in a polypeptide in the present invention. The NetNglyc server predicts N- Glycosylation sites in proteins using artificial neural networks that examine the sequence context of Asn- Xaa-Ser/Thr sequons. The NetNGlyc 1.0 and NetOGlyc 3.1 Prediction software can be accessed at the EXPASY website. In one embodiment, N-glycosylation occurs in the target antigen polypeptide of the fusion polypeptide described herein. In another embodiment, N-glycosylation occurs in the LFn polypeptide of a fusion polypeptide described herein, for example, at asparagine positions 62, 212, and/or 286, all of which have the potential of > 0.51 according to the NetNGlyc 1.0 Prediction software. Various combinations of N-glycosylation in the fusion polypeptide of the present invention are possible. In some embodiments, the individual and fusion polypeptides described herein have a single N-glycosylation at one of these three sites: asparagine positions 62, 212, and 286 of LFn. In other embodiments, the individual and fusion polypeptides described herein are N-glycosylated at two of these three sites: asparagine positions 62, 212, and 286 of LFn. In another embodiment, the individual and fusion polypeptides described herein is N-glycosylated at all three sites: asparagine positions 62, 212, and 286 of LFn. In yet another embodiment, N-glycosylation occurs in both the target antigen polypeptide (NP, Ml, and M2) and the LFn polypeptide. In some embodiments, the glycans of the individual and fusion polypeptide described herein are modified, for example, sialyated or asialyated. Glycosylation analysis of proteins is known in the art, for example, via glycan hydrolysis (using enzymes such as N-glycosidase F, EndoS endoglycosidase, sialidase or with 4N trifluroacetic acid), derivitization, and chromatographic separation such as LC-MS or LC-MS/MS (Pei Chen et. al., 2008, J. Cancer Res. Clin.Oncology, 134: 851-860; Kainz,E. et. al., 2008, Appl. Environ. Microbiol., 74: 1076-1086). LFn is predicted to have no O-linked glycosylation sites of > 0.50 potential.

[0117] In one embodiment, the intact cell is a living cell with an unbroken, uncompromised plasma membrane. A living cell would generally have a defined differential membrane potential across the membrane, with the inside of the cell being negative with respect to the outside of the cell. In one embodiment, the intact cell is a mammalian cell, including, for example, an antigen-presenting cell. [0118] While the whole of the N-terminal amino acid residues 1-288 (i. e. domain I of crystal structure, Pannifer et. al., 2001, Nature 414:229-233) of the LF polypeptide promotes the transmembrane delivery of other proteins, it should be understood that smaller fragments of domain I can be sufficient to translocate across cell membrane and promote the transmembrane delivery of other proteins, e. g., when fused together as a fusion polypeptide. The x-ray crystal structure of domain I shows 12 alpha helices and four beta sheet secondary protein structures (Pannifer et. al., 2001, supra). Smaller fragments of domain I that preserve these alpha helices and/or beta sheet secondary protein structures of domain I can translocate across cell membrane and promote the transmembrane delivery of other proteins when fused together as a fusion polypeptide. One skilled in the art can determine the presence of alpha helices and beta sheet secondary protein structure in the LFn polypeptide of the fusion polypeptide using methods known in the art, such as circular dichroism (CD).

[0119] In one embodiment, the LFn polypeptide of a vaccine composition described herein comprises at least the 60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists essentially of 60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists of 60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. [0120] In one embodiment, the LFn polypeptide of a vaccine composition described herein comprises at least the 80 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists essentially of 80 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists of 80 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. [0121] In one embodiment, the LFn polypeptide of a vaccine composition described herein comprises at least the 104 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists essentially of 104 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists of 104 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof. [0122] In one embodiment, the LFn polypeptide of a vaccine composition described herein comprises the amino acid sequence corresponding to SEQ. ID. No. 5, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists essentially of the amino acid sequence corresponding to SEQ. ID. No. 5, or a conservative substitution variant thereof. In one embodiment, the LFn polypeptide of a vaccine composition described herein consists of the amino acid sequence corresponding to SEQ. ID. No. 5, or a conservative substitution variant thereof. [0123] In one embodiment, the LFn polypeptide of a vaccine composition described herein comprises the amino acid sequence corresponding to SEQ. ID. No. 4, or a conservative substitution variant thereof. In another embodiment, the LFn polypeptide of a vaccine composition described herein consists essentially of the amino acid sequence corresponding to SEQ. ID. No. 4, or a conservative substitution variant thereof. In yet another embodiment, the LFn polypeptide of a vaccine composition described herein consists of the amino acid sequence corresponding to SEQ. ID. No. 4, or a conservative substitution variant thereof.

[0124] In one embodiment, the LFn polypeptide of a vaccine composition described herein comprises the amino acid sequence corresponding to SEQ. ID. No. 3, or a conservative substitution variant thereof. In another embodiment, the LFn polypeptide of a vaccine composition described herein consists essentially of the amino acid sequence corresponding to SEQ. ID. No. 3, or a conservative substitution variant thereof. In yet another embodiment, the LFn polypeptide of a vaccine composition described herein consists of the amino acid sequence corresponding to SEQ. ID. No. 3, or a conservative substitution variant thereof.

[0125] In one preferred embodiment, the LFn polypeptide of a vaccine composition described herein, promotes transmembrane delivery.

[0126] In one embodiment, the LFn polypeptide of a vaccine composition described herein, the LFn polypeptide does not bind B. anthracis protective antigen (PA) protein. The PA protein is the natural binding partner of LF, forming bipartite protein exotoxin, lethal toxin (LT). The PA protein is a 735- amino acid polypeptide, a multi-functional protein that binds to cell surface receptors, mediates the assembly and internalization of the complexes, and delivers them to the host cell endosome. Once PA is attached to the host receptor, it is cleaved by a host cell surface (furin family) protease before it is able to bind LF. The cleavage of the N-terminus of PA enables the C-terminal fragment to self-associate into a ring-shaped heptameric complex (prepore) that can bind LF and delivers LF into the cytosol. The N- terminal fragment (residues 1-288, domain I) can be expressed as a soluble folded domain that maintains the ability to bind PA and enables the translocation of heterologous fusion proteins into the cytosol. Smaller fragments of this residue 1-288 N-terminal fragment have been shown to also translocate heterologous fusion proteins into the cytosol in the absence of PA. Hence, in one embodiment, smaller fragments described herein can translocate across membranes but do not bind PA. [0127] In one embodiment, the LFn polypeptide of a vaccine composition described herein, the LFn polypeptide substantially lacks amino acids 1-33 of SEQ. ID. No. 3. Amino acids 1-33 of SEQ. ID. No. 3 encompass the signal peptide that is predicted to direct the post-translational transport of the LF protein. In some embodiments, the LFn polypeptide of any of the fusion polypeptides described herein lacks a signal peptide that functions to direct the post-translational transport of the fusion polypeptide. In other embodiments, the LFn polypeptide of the fusion polypeptides described herein comprises a signal peptide for co-translation on the ER. The signal peptide is also called a leader peptide in the N- terminus, which may or may not be cleaved off after the translocation through the ER membrane. One example of a signal peptide is MAPFEPLASGILLLLWLIAPSRA (SEQ. ID. No. 17). Other examples of signal peptides can be found at SPdb, a Signal Peptide Database, which is found at the world wide web site of http colon "forward slash" "forward slash" proline "period" bic "period" nus "period" edu "period" sg "forward slash" spdb "forward slash".

[0128] In one embodiment, the vaccine compositions described herein further comprise an adjuvant. Examples of adjuvants include, but are not limited to QS- 21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

[0129] In one embodiment of a vaccine composition described herein, the influenza virus is selected from strains consisting of influenza virus A HlNl A/Sw/Iowa/31 , H2N3 A/Mal/Al/77, H2N9 A/Pintail/Alb/293/77, H3N2 A/Ty/Eng/69, H3N8 A/Dk/Ukraine/1/63, H4N8 A/Dk/Czech/56, H5N1 A/Ck/Scot/59, H5N2 A/Quail/Ore/20719/86, H5N2 A/Ck/Wa/13413/84, H5N2 A/Ck/Penn/13701/83, H5N2 A/Ty/Min/3689-1551/81, H5N3 A/Ty/cA/35621/84, H5N3 A/Tern/SA/61, H5N8 A/Ty/Ireland/83, H5N9 A/Ty/Wis/68, H5N9 A/Ty/Ont/7732/66, H5N9 A/Ck/Que/14588-19 (Mex. Isolate), H5N2 A/Ck/Hidalgo/26654-1368/94 (Mex. Isolate), H5N2 A/Ck/Pue/8623-607 (Mex. Isolate), H5N? A/Emu/Tx/39924/93, H5N3 A/Emu/Tx/39924/93 (IB clone E2), H6N2 Field Isolate, Cnn00053, H6N8 A/Ty/Ont/63, H7N2 A/Ty/Ore/71, H7N3 A/Ck/Aust/3634/92, H7N3 A/Ty/MN/29206/83, H7N7 A/Ck/Vic/32972/85, H7N8 A/Magrob/China/28710/93, H7N9 A/Ty/MN/38429/88, H8N4 A/Ty/Ont/61181/67, H9N2 A/Ty/MN/12877/1285/81, H9N2 A/Ty/Wis/1/66, H9N9 A/Pheasent/Wa/37, H10N7 A/Ck/Germany/49, H10N8 A/Quail/Ithaca/1117/65, HI lNl A/Dk/Eng/56, H11N9 A/Dk/Memphis/546/74, H12N1 A/Dk/Alberta/60/76, H13N1 A/Gull/MD/704/77, H14N5 A/Mal/Gurjev/263/83, Hl, H1N2, , influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, B/Shanghai, B/Paris, and B/Hong Kong.

[0130] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0- 911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); The ELISA guidebook (Methods in molecular biology 149) by Crowther J. R. (2000); Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis, 1979, Scientific Newsletters; Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0131] Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987)); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.); Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.); Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), and Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entirety. [0132] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0133] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean ±1%.

[0134] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [0135] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Definitions of terms

[0136] The term "vaccine composition" used herein is defined as composition used to elicit an immune response against an antigen within the composition in order to protect or treat an organism against disease.

[0137] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. [0138] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. [0139] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0140] As used herein, the term "fused" means that one protein is physically associated with a second protein, for example via an electrostatic or hydrophobic interaction or a covalent linkage. Covalent linkage can encompass linkage as a fusion protein or chemically coupled linkage, for example via a cysteine residue.

[0141] As used herein, the term "fusion polypeptide" or "fusion protein" means a protein created by joining two polypeptide coding sequences together. The fusion polypeptides of this invention are fusion polypeptides formed by joining a coding sequence of a LF polypeptide or fragment or mutant thereof with a coding sequence of a second polypeptide to form a fusion or chimeric coding sequence such that they constitute a single open-reading frame. The fusion coding sequence, when transcribed and translated, expresses a fusion polypeptide. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins which are joined by a peptide bond. [0142] As used herein, the term "protein" and "polypeptide" are used interchangeably. [0143] As used herein, the term "promotes transmembrane delivery" refers to the ability of a first polypeptide to facilitate a second protein to traverse the membrane of an intact, living cell. [0144] As used herein, the term "cytosol" refers to the interior of an intact cell. The "cytosol" comprises the cytoplasm and the organelles inside a cell.

[0145] As used herein, the term "an intact cell " refers to a living cell with an unbroken, uncompromised plasma membrane, which cell has a differential membrane potential across the membrane, with the inside of the cell being negative with respect to the outside of the cell. [0146] As used herein, the term "N-glycosylated" or "N-glycosylation" refers to the covalent attachment of a sugar moiety to asparagine residues in a polypeptide. Sugar moieties can include but are not limited to glucose, mannose, and N-acetylglucosamine. Modifications of the glycans are also included, e. g. siaylation. The LFn polypeptide has three N-glycosylation sites: asparagine positions 62, 212, and 286 in the 809 amino acid polypeptide.

[0147] As used herein, the terms "N-glycosylated LFn-fusion polypeptide," "N-glycosylated LF-fusion polypeptide" or "N-glycosylated fused polypeptide" refer to a fusion polypeptide, as defined herein, that has at least one sugar moiety covalently attached to an asparagine residue. For example, Asn-62, Asn- 212, and Asn-286 can be glycosylated in an N-glycosylated LF-fusion polypeptide. [0148] As used herein, the term "substantially lacks amino acids 1-33" in the context of a fusion polypeptide described herein refers to a fusion polypeptide that lacks signal peptide activity. [0149] As used herein, the term "antigen" refers to any substance that prompts an immune response directed against the substance.

[0150] An antigen presenting cell is a cell that expresses the Major Histocompatibility complex (MHC) molecules and can display foreign antigen complexed with MHC on its surface. Examples of antigen presenting cells are dendritic cells, macrophages, B cells, fibroblasts (skin), thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells. [0151] The term "lethal factor" or "LF" as used herein refers generally to a non-PA polypeptide of the bipartite B. anthracis exotoxin. Wild-type, intact B. anthracis LF polypeptide has the amino acid sequence set out in GenBank Accession Number M29081 (Gene ID No: 143143), which corresponds to SEQ ID NO: 1. SEQ ID NO: 1 corresponds to LF with a signal peptide located at residues 1 to 33 at its N-terminus. Stated another way, immature wild-type LF corresponds to an 809 amino acid protein, which includes a 33 amino acid signal peptide at the N-terminus. The amino acid sequence of immature wild- type LF (SEQ ID NO: 1) with the signal peptide highlighted in bold is as follows:

[0152] MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKR

KDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDI

TKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGK

ILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAK

AFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLSRYEKWEKIKQHYQHWS

DSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSE

EEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQ

NIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNY

MIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVV PKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITFNVHNRYASNIVESAYLILNEWKNNIQSDLIK

KVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDS

EGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHS

TDHAERLKVQKNAPKTFQFINDQIKFIINS (SEQ ID NO: 1)

[0153] Cleavage of the immature LF protein results in a mature wild-type LF polypeptide of 776 amino acids in length. The 776 amino acid polypeptide sequence of mature wild-type LF polypeptide (i.e. lacking the N-terminal signal peptide) corresponds to SEQ ID NO: 2, as follows:

[0154] AGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKE

AAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAK

EGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDL

LFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEI

NLSLEELKDQRMLSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKE

LLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQP

YDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLG

ADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLEN

GKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLI

TFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEI

YEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKK

FIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS (SEQ ID NO: 2).

[0155] The term "LF polypeptide" applies not only to full-length, wild-type LF (with or without the signal sequence), but also to fragments thereof that mediate intracellular delivery of fused or physically associated polypeptides to an intact cell, such as, an antigen presenting cell. Also included in the term "LF polypeptide" are conservative substitution variants of LF, including conservative substitution variants that mediate such intracellular delivery.

[0156] The term "LFn polypeptide" refers to an N-terminal fragment of B. anthracis LF that does not display zinc metalloproteinase activity and does not inactivate mitogen-activated kinase activity, yet does mediate intracellular or transmembrane delivery of fused polypeptides. LFn polypeptides as defined and described herein are preferred. In one aspect, "LFn polypeptide" includes SEQ ID NO: 3, which corresponds to a 288 amino acid immature LFn protein; this LFn protein is "immature" in that it includes a signal peptide located at residues 1 to 33 of the N-terminus. Stated another way, immature LFn corresponds to a 288 amino acid protein, which includes a 33 amino acid signal peptide at the N- terminus. Cleavage of the immature LFn protein of SEQ ID NO: 3 results in a mature LFn polypeptide of 255 amino acids in length. It should be emphasized that, for the purposes of the methods and compositions described herein, the LF and/or LFn polypeptides can either include or lack the signal peptide - that is, the presence or absence of the signal peptide is not expected to influence the activity of LF polypeptides as transmembrane transport facilitators in the methods described herein. The amino acid sequence of immature LFn (SEQ ID NO: 3) with the signal peptide highlighted in bold is as follows: [0157] MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKD

EERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITK

HISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKIL

SRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKA

FAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLS (SEQ ID NO: 3)

[0158] The polypeptide sequence of a mature LFn polypeptide (which lacks the N-terminal signal peptide) is 255 amino acids in length and corresponds to SEQ ID NO: 4 is as follows:

[0159] AGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKE AAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAK EGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDL LFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEI NLS (SEQ ID NO: 4)

[0160] The term "functional fragment" as used in the context of a "functional fragment of LFn" refers to a fragment of an LFn polypeptide that mediates, effects or facilitates transport of an antigen across an intact, living cell's membrane. One example of such a fragment of an LFn polypeptide is a 104 amino acid C-terminal fragment of LFn corresponding to SEQ ID NO: 5 as follows (this sequence is also disclosed as SEQ ID NO: 3 in U.S. Patent Application 10/473190, which is incorporated herein by reference):

[0161] GKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSN EVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLS (SEQ ID NO: 5) [0162] The term "LFn polypeptide" as used herein encompasses each of the "immature" LFn and "mature" LFn nolecules described herein, as well as fragments, variants (including conservative substitution variants) and derivatives thereof that mediate, effect or facilitate transport of a physically associated, e.g., fused, polypeptide across the membrane of an intact, living cell. Additional fragments of LFn polypeptides specifically contemplated for use in the methods, compositions and kits described herein include a fragment comprising, or optionally, consisting essentially of the C-terminal 60, 80, 90, 100 or 104 amino acids of SEQ ID NO: 3 or a conservative substitution variant thereof that mediates, effects or facilitates transfer of a physically associated, e.g., fused polypeptide across an intact membrane of a living cell.

[0163] The term "NP" refers to the Influenza A virus (A/Paris/908/97(H3N2)) nucleoprotein (NP) polypeptide having the amino acid sequence encoded by the NP cDNA set out in Genbank Accession No. AF483604 (Gene ID: GL21902317). The amino acid sequence (Genbank Accession No. AAM78513.1 (Gene ID: GI: 21902318)) is as follows:

MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIEK MVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYKRVDGRWMRELVLYDKEEIRRIWRQANNGED ATAGLTHMMIWHSNLNDTTYQRTRAL VRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTM VMELIRMVKRGINDRNFWRGENGRKTRSAYERMCNILKGKFQTAAQRAMVDQVRESRNPGNA EIEDLIFLARSALILRGSVAHKSCLP ACVYGP AVSSGYDFEKEGYSLVGIDPFKLLQNSQVYSLIRP NENP AHKSQL VWMACHSAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDNMGSSTLELR SGYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFEKSTVMAAFTGNTEGRTSDMRAEIIR MMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDN (SEQ. ID. NO: 6). The term also refers to naturally occurring NP variants from different species of influenza viruses, virus subtypes and strains, for example, the NPs from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C. Examples of virus subtypes include, but are not limited to Influenza virus A HlNl, which caused Spanish flu in 1918, Influenza virus A H2N2, which caused Asian Flu in 1957, Influenza virus A H3N2, which caused Hong Kong Flu in 1968, Influenza virus A H5N1, a pandemic threat in the 2007-08 flu season, Influenza virus A H7N7, which has unusual zoonotic potential, Influenza virus A H1N2, endemic in humans and pigs, Influenza virus A H9N2, Influenza virus A H7N2, Influenza virus A H7N3, Influenza virus A H10N7, B/Victoria/2/87, B/Hong Kong/1351/02, B/Shanghai/361/2002 and B/Yamagata/16/88. The NP variants from virus A are at least 85% homologous to SEQ. ID. No: 6. The NP variants from viruses B and C are at least 35% homologous to SEQ. ID. No: 6 at the aligned segments using conventional sequence alignment software known in the art and described herein.

[0164] The term "Ml" refers to the Influenza A virus (A/chicken/Chile/4977/02(H7N3)) matrix protein 1, which has the amino acid sequence set out in Genbank Accession No. AY303656 (Gene ID: GI: 34597766). The amino acid sequence (Genbank Accession No. AAQ77440.1 (Gene ID: GI: 34597767)) is as follows:

MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTL TVPSERGLQRRRFVQNALNGNGDPNNMDRAVKL YRKLKREITFHGAKEV ALSYSTGALASCMG LIYNRMGTVTTEV AFGLVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTT AKAMEQM AGSSEQAAEAMEVASQARQMVQAMRTIGTHPSSSAGLKDDLLENLQAYQKRMGVQMQRFK (SEQ. ID. No. 7). The term also refers to naturally occurring Ml variants from different species of influenza viruses, virus subtypes and strains. For example, the MIs from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C. Examples of virus subtypes include but are not limited to Influenza virus A HlNl, which caused Spanish flu in 1918, Influenza virus A H2N2, which caused Asian Flu in 1957, Influenza virus A H3N2, which caused Hong Kong Flu in 1968, Influenza virus A H5N1, a pandemic threat in the 2007-08 flu season, Influenza virus A H7N7, which has unusual zoonotic potential, Influenza virus A H1N2, endemic in humans and pigs, Influenza virus A H9N2, Influenza virus A H7N2, Influenza virus A H7N3, Influenza virus A H10N7, B/Victoria/2/87, B/Hong Kong/1351/02, B/Shanghai/361/2002 and B/Yamagata/16/88. The Ml variants from virus A are at least 85% homologous to SEQ. ID. No. 7. The Ml variants from viruses B and C are at least 35% homologous to SEQ. ID. No. 7 at the aligned segments using conventional sequence alignment software known in the art and described herein.

[0165] The term "M2" refers to the Influenza A virus (A/chicken/Chile/4977/02(H7N3)) matrix protein 2, whose has the amino acid sequence is encoded by the M gene set out in Genbank Accession No. AY303656 (Gene ID: GI: 34597766). The amino acid sequence (Genbank Accession No. AAQ77441.1 (Gene ID: GI: 34597768)) is as follows:

MSLLTEVETPTRNGWECKCSDSSDPL VIAASIIGILHLILWILDRLFFKCIYRRLKYGLKRGPSTEG VPESMREEYRQEQQSAVDVDDSHFVNIELE (SEQ. ID. NO. 8). The term also refer to naturally occurring M2 variants from different species of influenza viruses, virus subtypes and strains. For example, the M2s from the three species of influenza virus that infects humans: Influenza virus A, Influenza virus B, and Influenza virus C. Examples of virus subtypes include but are not limited to Influenza virus A HlNl, which caused Spanish flu in 1918, Influenza virus A H2N2, which caused Asian Flu in 1957, Influenza virus A H3N2, which caused Hong Kong Flu in 1968, Influenza virus A H5N1, a pandemic threat in the 2007-08 flu season, Influenza virus A H7N7, which has unusual zoonotic potential, Influenza virus A H1N2, endemic in humans and pigs, Influenza virus A H9N2, Influenza virus A H7N2, Influenza virus A H7N3, Influenza virus A H10N7, B/Victoria/2/87, B/Hong Kong/1351/02, B/Shanghai/361/2002 and B/Yamagata/16/88. The M2 variants from virus A are at least 85% homologous to SEQ. ID. No. 8. The M2 variants from viruses B and C are at least 35% homologous to SEQ. ID. No. 8 at the aligned segments using conventional sequence alignment software known in the art and described herein.

[0166] The term "HA" refers to hemagglutinin subtype H5 of Influenza A virus (A/Hong Kong/156/97(H5N1))], which has the amino acid sequence set out in Genbank Accession No. AAC32088.1, GL3335421. The amino acid sequence (Genbank Accession No. AAC32088.1, GL3335421 is as follows:

MEKTVLLLATVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILERTHNGKLCDLNGVK PLILRDCSV AGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGNFNDYEELKHLLSRINHFEK IQIIPKSSWSNHDASSGVSSACPYLGRSSFFRNV VWLIKKNSAYPTIKRS YNNTNQEDLLVLWGIH HPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPEIATRPKVNGQSGRMEFFWTILKPNDAINFESN GNFIAPEY AYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSN RLVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIINKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN ERTLDFHDSNVKNL YDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYD YPQYSEEA

RLNREEISGVKLESMGTYQILSIYSTVASSLALAIMV AGLSLWMCSNGSLQCRICI (SEQ. ID. NO. 25). The term also refers to naturally occurring and genetically engineered HA variants from different species of influenza viruses, virus subtypes and strains. For example, the HAs from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C. [0167] The term "NA" refers to neuraminidase subtype 1 of Influenza A virus (A/Hong Kong/156/97(H5N1)), which has the amino acid sequence encoded by the RNA segment 6 set out in Genbank Accession No. AF046089.1, GL3335422. The amino acid sequence (Genbank Accession No. AAC32089.1, GL3335423 is as follows:

MNPNQKIITIGSICMVVGIISLMLQIGNIISVWVSHIIQTWHPNQPEPCNQSINFYTEQAAASVTLA GNSSLCPISGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRS PYRTLMSCPVGEAPSPYNSRFESVAWSASACHDGISWLTIGISGPDNGAV AVLKYNGIITDTIKS WRNNILRTQESECACVNGSCFTVMTDGPSNEQASYKIFKIEKGRVVKSVELNAPNYHYEECSCY PDAGEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDSPRPNDGTGSCGPVSLNGA YGV KGFSFKYGNGVWIGRTKSTSSRSGFEMIWDPNGWTETDSSFSLKQDIIAITDWSGYSGSFIQHPEL TGLNCMRPCFWVELIRGRPKEKTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK (SEQ. ID. No. 26). The term also refers to naturally occurring and genetically engineered NA variants from different species of influenza viruses, virus subtypes and strains. For example, the NAs from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C.

[0168] The term "NSl" refers to the nonstructural protein 1 of the Influenza A virus (A/Hong Kong/1073/99(H9N2)), which has the amino acid sequence encoded by RNA segment 8 set out in Genbank Accession No. NC_004906.1, GI:32140160. The amino acid sequence (Genbank Accession No. NP_859034.1, GI:32140161 is as follows:

MDSNTVSSFQVDCFLWHVRKRFADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIRTATREGKHI VERILEEESDEALKMTIASVPASRYLTEMTLEEMSRDWLMLIPKQKVTGPLCIRMDQAVMGKTII LKANFSVIFNRLEALILLRAFTDEGAIVGEISPLPSLPGHTDEDVKNAIGVLIGGLEWNDNTVRVS

ETLQRFTWRSSDENGRSPLPPKQKRKVERTIEPEV (SEQ. ID. NO. 27). The term also refers to naturally occurring NSl variants from different species of influenza viruses, virus subtypes and strains.

For example, the NSIs from the three species of influenza virus that infect humans: Influenza virus A,

Influenza virus B, and Influenza virus C.

[0169] The term "NS2" refers to the nonstructural protein 2 of the Influenza A virus (A/Hong

Kong/1073/99(H9N2)), which has the amino acid sequence encoded by the RNA segment 8 set out in

Genbank Accession No. NC_004906.1, GI:32140160. The amino acid sequence (Genbank Accession No.

YP_581750.1, GI:93211154 is as follows:

MDSNTVSSFQDILTRMSKMQLGSSSEDLNGMITQFESLKLYRDSLGEAVMRMGDLHSLQNRNG

KWREQLSQKFEEIRWLIEEMRHRLRITENSFEQITFMQALQLLLEVEQEIRTFSFQLI (SEQ. ID. NO.

28). The term also refers to naturally occurring NS2 variants from different species of influenza viruses, virus subtypes and strains. For example, the NS2s from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C.

[0170] The term "PB 1" refers to the RNA-directed RNA polymerase catalytic subunit of the RNA - dependent RNA polymerase of Influenza A virus (A/Hong Kong/485/97(H5Nl)), which has the amino acid sequence encoded by segment 2 set out in Genbank Accession No. AF084266.1, GI: 8307774. The amino acid sequence (Genbank Accession No. AAF74316.1, GI:8307775 is as follows:

MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGRWTTNTETGA

PQLNPIDGPLPEDNEPSGYAQTDCVLEAMAFLEESHPGLFENSCLETMEVVQQTRVDKLTQGRQ

TYDWTLNRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKEEMEITTHFQRKRRV

RDNMTKKMVTQRTIGKKKQRLTKKSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVHF

VEALARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTVTGDNTKWNENQNPRI

FLAMITYITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFESKSMKLRTQIPAEMLANIDLKYFN ESTRTKIEKIRPLLVEGTASLSPGMMMGMFNMLSTVLGVSILNLGQKKYTKTTYWWDGLQSSD DF ALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINRTGTFEFTSFFYRYGFV ANFSMELP SFGVSGINESADMSIGVTVIKNNMINNDLGP ATAQMALQLFIKD YR YTYRCHRGDTQIQTRRSFE LKKLWEQTRSKAGLLVSDGGPNL YNIRNLHIPEVCLKWELMDEDYQGRLCNPLNPFVSHKEVE SVNNA VVMPAHGPAKSMEYDAV ATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCTLFEKFFPS SSYRRPVGISSMMEAMVSRARID ARIDFESGRIKKEEFAEILKICSTIEELGRQGK (SEQ. ID. NO. 29). The term also refers to naturally occurring PBl variants from different species of influenza viruses, virus subtypes and strains. For example, the PBIs from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C. [0171] The term "PB2" refers to the polymerase basic protein 2 of Influenza A virus (A/HongKong/485/97(H5Nl)), which has the amino acid sequence encoded by segment 1 set out in Genbank Accession No. AF084263.1, GI:8307768. The amino acid sequence (Genbank Accession No. AAF74313.1, GL8307769 is as follows:

MERIKELRDLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRI

MEMIPERKEQGQTLWSKTNDAGSDRVMVSPLAVTWWNRNGPTTSTVHYPKVYKTYFEKVERL

KHGTFGPVHFRNQVKIRRRVDMNPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEK

REELKNCNIAPLMV AYMLERELVRKTRFLPV AGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRN

DDVDQSLIIAARNIVRRATVSADPLVSLLEMCHSTQIGGVRMVDILKQNPTEEQAVDICKAAMG

LKISSSFSFGGFTFKRTKGFSVKREEEVLTGNLQTLKIKVHEGYEEFTMVGRRATAILRKATRRMI

QLIVSGRDEQSIAEAIIV AMVFSQEDCMIKA VRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLF

QNWGIEPIDNVMGMIGILPDMTPSTEMSLRGVRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGN

VLLSPEEVSETQGMEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETVKIQWSQEPTMLYNK

MEFEPFQSLVPKAARSQYSGFVRTLFQQMRDVLGTFDTVQIIKLLPFAAAPPKQSRMQFSSLTVN

VRGSGMRILVRGNSPAFNYNKTTKRLTILGKDAGALTEDPDEGTAGVESAVLRGFLILGKEDKR

YGPALSINELSNLTKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN (SEQ. ID. NO. 30). The term also refers to naturally occurring PB2 variants from different species of influenza viruses, virus subtypes and strains. For example, the PB2s from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C.

[0172] The term "PA," when used in reference to influenza virus, refers to the polymerase acidic protein of Influenza A virus (A/HongKong/485/97(H5Nl)), which has the amino acid sequence encoded by segment 3 set out in Genbank Accession No. AF084270.1, GI: 8307782. The amino acid sequence (Genbank Accession No. AF084270.1, GI:8307782 is as follows:

MEDFVRQCFNPMIVELAEKTMKEYGEDPKIETNKFAAICTHLEVCFMYSDFHFIDERGESIIVESG DPNALLKHRFEIIEGRDRAMAWTVVNSICNTTGVDKPKFLPDLYDYKENRFTEIGVTRREIHIYY LEKANKIKSEKTHIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMASRGLWDSFRQSERG EETIEERFEITGTMRRLADQSLPPNFSSLENFRA YVDGFKPNGCIEGKLSQMSKEVNARIEPFLKT TPRPLRLPDGPPCSQRSKFLLMD ALKLSIEDPSHEGEGIPL YD AIKCMKTFFGWREPNIIKPHEKGI NPNYLMAWKQVLAELQDIENEDKIPKTKNMKKTSQLMWALGENMAPEKVDFEDCKDIDDLKQ YHSDEPELRSLASWIQSEFNKACELTDSSWIELDEIGEDV APIEHIASMRRNYFT AEVSHCRATEY IMKGVYINT ALLNASCAAMDDFQLIPMISKCRTKEGRRKTNL YGFIIKGRSHLRNDTD VVNFVS MEFSLTDPRLEPHKWEKYCVLEIGEMLLRTAIGQVSRPMFL YVRTNGTSKIKMKWGMEMRRCL LQSLQQIESMIEAESSIKEKDMTKEFFENRSETWPIGESPKGVEEGSIGKVCRTLLAKSVFNSLYSS PQLEGFS AESRKLLLIVQALRDNLEPGTFDLEGL YGAIEECLINDPWVLLNASWFNSFLTHALR (SEQ. ID. No. 31). The term also refers to naturally occurring PA variants from different species of influenza viruses, virus subtypes and strains. For example, the PAs from the three species of influenza virus that infect humans: Influenza virus A, Influenza virus B, and Influenza virus C. [0173] The term "adjuvant" as used herein refers to any agent or entity which increases the antigenic response or immune response by a cell to a target antigen. Examples of adjuvants include, but are not limited to mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; other peptides; oil emulsions; and potentially useful human adjuvants such as BCG and Corynebacterium parvum. QS- 21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL- 1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

[0174] The terms "protective antigen" or "PA" (when used relative to B. anthracis) are used interchangeably herein to refer to part of the B. anthracis exotoxin bipartite protein which binds to a mammalian cell's surface by cellular receptors. A "PA," as the term is used in this context has its receptor binding site intact and functional. U. S. Patent Nos. 5,591,631 and 5,611 ,21 A (incorporated by reference in their entirety) describe PA fusion proteins that target PA to particular cells, such as cancer cells and HIV-infected cells, using as fusion partners ligands for receptors on the targeted cells. [0175] A "fragment" of a target antigen as that term is used herein will be at least 6 amino acids in length, and can be, for example, at least 8, at least 10, at least 14, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 25 amino acids or greater.

[0176] The terms "Cytotoxic T Lymphocyte" or "CTL" refers to lymphocytes which induce apoptosis in targeted cells. CTLs form antigen-specific conjugates with target cells via interaction of TCRs with processed antigen (Ag) on target cell surfaces, resulting in apoptosis of the targeted cell. Apoptotic bodies are eliminated by macrophages. The term "CTL response" is used to refer to the primary immune response mediated by CTL cells.

[0177] The term "cell mediated immunity" or "CMI" as used herein refers to an immune response that does not involve antibodies or complement but rather involves the activation of, for example, macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes (T -cells), and the release of various cytokines in response to a target antigen. Stated another way, CMI refers to immune cells (such as T cells and lymphocytes) which bind to the surface of other cells that display a target antigen (such as antigen presenting cells (APS)) and trigger a response. The response may involve either other lymphocytes and/or any of the other white blood cells (leukocytes) and the release of cytokines. Cellular immunity protects the body by: (i) activating antigen-specific cytotoxic T-lymphocytes (CTLs) that are able to destroy body cells displaying epitopes of foreign antigen on their surface, such as virus- infected cells and cells with intracellular bacteria; (2) activating macrophages and NK cells, enabling them to destroy intracellular pathogens; and (3) stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

[0178] The term "immune cell" as used herein refers to any cell which can release a cytokine in response to a direct or indirect antigenic stimulation. Included in the term "immune cells" herein are lympocytes, including natural killer (NK) cells, T-cells (CD4+ and/or CD8+ cells), B-cells, macrophages and monocytes, Th cells; ThI cells; Th2 cells; Tc cells; leukocytes; dendritic cells; macrophages; mast cells and monocytes and any other cell which is capable of producing a cytokine molecule in response to direct or indirect antigen stimulation. Typically, an immune cell is a lymphocyte, for example a T-cell lymphocyte.

[0179] The term "cytokine" as used herein is used interchangeably with the term "effector molecule," and refers to a molecule released from an immune cell in response to stimulation with an antigen.

Examples of such cytokines include, but are not limited to: GM-CSF; IL-lα; IL-I β; IL -2; IL-3; IL -4; IL-

5; IL-6; IL-7; IL-8; IL-10; IL-12; IFN-OC; IFN-β; IFN-γ; MIP-Ia; MlP-lβ; TGF-β; TNFα and TNFβ. The term "cytokine" does not include antibodies.

[0180] The term "complex" as used herein refers to a collection of two or more molecules, connected spatially by means other than a covalent interaction; for example they can be connected by electrostatic interactions such as van der Waals forces etc.

[0181] The term "translocated into a cell" refers to the movement of a moiety, such as a target antigen, and optionally a fusion polypeptide described herein from a location outside a cell, across the plasma membrane to the inside of an intact, living cell.

[0182] The term "in vivo" refers to assays or processes that occur in an animal.

[0183] The term "mammal" is intended to encompass a singular "mammal" and plural "mammals," and includes, but is not limited to humans; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and bears. In some embodiments, a mammal is a human.

[0184] The term "pharmaceutically acceptable" refers to compounds and compositions which may be administered to mammals without undue toxicity. The term "pharmaceutically acceptable carriers" excludes tissue culture medium. Exemplary pharmaceutically acceptable salts include but are not limited to mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like, and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.

[0185] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues linked by peptide bonds, and for the purposes of the claimed invention, have a minimum length of at least 15 amino acids. Oligopeptides, oligomers multimers, and the like, typically refer to longer chains of amino acids and are also composed of linearly arranged amino acids linked by peptide bonds, whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non-naturally occurring amino acids, are included within this definition. Both full- length proteins and fragments thereof greater than 15 amino acids are encompassed by the definition. The terms also include polypeptides that have co-translational (e.g., signal peptide cleavage) and post- translational modifications of the polypeptide, such as, for example, disulfide -bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like. Furthermore, as used herein, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods. For the methods and compositions described herein, the term "peptide" refers to a sequence of peptide bond-linked amino acids containing between 6 amino acids and 15 amino acids in length.

[0186] It will be appreciated that proteins or polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, can be modified in a given polypeptide, either by natural processes such as glycosylation and other post-translational modifications, or by chemical modification techniques which are well known in the art. Known modifications which can be present in polypeptides of the present invention include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formulation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0187] As used herein, the terms "homologous" or "homologues" are used interchangeably, and when used to describe a polynucleotide or polypeptide, indicate that two polynucleotides or polypeptides, or designated sequences thereof, when optimally aligned and compared, for example using BLAST, version 2.2.14 with default parameters for an alignment (see herein) are identical, with appropriate nucleotide insertions or deletions or amino-acid insertions or deletions, in at least 70% of the nucleotides, usually from about 75% to 99%, and more preferably at least about 98 to 99% of the nucleotides. For a polypeptide, there should be at least 50% of amino acid identity in the polypeptide. The term "homolog" or "homologous" as used herein also refers to homology with respect to structure. Determination of homologs of genes or polypeptides can be easily ascertained by the skilled artisan. When in the context with a defined percentage, the defined percentage homology means at least that percentage of amino acid similarity. For example, 85% homology refers to at least 85% of amino acid similarity. [0188] As used herein, the term "heterologous" reference to nucleic acid sequences, proteins or polypeptides mean that these molecules are not naturally occurring in that cell. For example, the nucleic acid sequence coding for a fusion LFn-target antigen polypeptide described herein that is inserted into a cell, e. g. in the context of a protein expression vector, is a heterologous nucleic acid sequence. [0189] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0190] Where necessary or desired, optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981), which is incorporated by reference herein), by the homology alignment algorithm of Needleman and Wunsch (J. MoI. Biol. 48:443-53 (1970), which is incorporated by reference herein), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), which is incorporated by reference herein), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. (See generally Ausubel et al. (eds.), Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999)). [0191] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show the percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J. MoI. Evol. 25:351-60 (1987), which is incorporated by reference herein). The method used is similar to the method described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989), which is incorporated by reference herein). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. [0192] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described by Altschul et al. (J. MoI. Biol. 215:403- 410 (1990), which is incorporated by reference herein). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated by reference herein). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information internet web site. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990), supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992), which is incorporated by reference herein) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0193] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), which is incorporated by reference herein). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference amino acid sequence if the smallest sum probability in a comparison of the test amino acid to the reference amino acid is less than about 0.1, more typically less than about 0.01, and most typically less than about 0.001. [0194] The term "variant" as used herein refers to a polypeptide or nucleic acid that differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more specific functions or biological activities of the naturally occurring molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative," in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Substitutions encompassed by variants as described herein may also be "non conservative," in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties (e.g., substituting a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. Also encompassed within the term "variant," when used with reference to a polynucleotide or polypeptide, are variations in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild- type polynucleotide or polypeptide). A "variant" of an LFn polypeptide refers to a molecule substantially similar in structure and function to that of a polypeptide of SEQ ID NO: 3, where the function is the ability to mediate, effect or facilitate transport of an associated or fused polypeptide across a cell membrane of a living cell from a subject. In some embodiments, a variant of SEQ ID NO: 3 or SEQ ID NO: 4 is a fragment of SEQ ID NO: 3 or 4 as disclosed herein, such as SEQ ID NO: 5. [0195] The term "substantially similar," when used in reference to a variant of LFn or a functional derivative of LFn as compared to the LFn protein encoded by SEQ ID NO: 3 means that a particular subject sequence, for example, an LFn fragment or LFn variant or LFn derivative sequence, varies from the sequence of the LFn polypeptide encoded by SEQ ID NO: 3 by one or more substitutions, deletions, or additions relative to SEQ ID NO: 3, but retains at least 50% of the transmembrane transport facilitation activity, and preferably higher, e.g., at least 60%, 70%, 80%, 90% or more exhibited by the LFn protein of SEQ ID NO: 3. (It is acknowledged that LFn does not occur naturally - reference to a "native" or "natural" LFn sequence is intended to convey that the sequence is identical to the portion of naturally-occurring LF polypeptide designated as LFn herein.) In determining polynucleotide sequences, all subject polynucleotide sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference polynucleotide sequence, regardless of differences in codon sequence. A nucleotide sequence is "substantially similar" to a given LFn nucleic acid sequence if: (a) the nucleotide sequence hybridizes to the coding regions of the native LFn sequence, or (b) the nucleotide sequence is capable of hybridization to nucleotide sequence of LFn encoded by SEQ ID NO: 1 under moderately stringent conditions and has biological activity similar to the native LFn protein; or (c) the nucleotide sequences are degenerate as a result of the genetic code relative to the nucleotide sequences defined in (a) or (b). Substantially similar proteins will typically be greater than about 80% similar to the corresponding sequence of the native protein.

[0196] Variants can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Variants can also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. "Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); T) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984).)

[0197] The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and exposed to solvents, or on the interior and not exposed to solvents. Selection of such conservative amino acid substitutions is within the skill of one of ordinary skill in the art and is described, for example by Dordo et al., J. MoI Biol, 1999, 217, 721-739 and Taylor et al., J. Theor. Biol. 119(1986);205-218 and S. French and B. Robson, J. MoI. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent). These substitutions include, but are not limited to the following: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P. [0198] In alternative embodiments, one can also select conservative amino acid substitutions suitable for amino acids on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent). For example, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, LF polypeptides including non-conservative amino acid substitutions are also encompassed within the term "variants." A variant of an LFn polypeptide, for example a variant of SEQ ID NO: 3 or 4 is meant to refer to any molecule substantially similar in structure (i.e., having at least 50% homology as determined by BLASTp analysis using default parameters) and function (i.e., at least 50% as effective as a polypeptide of SEQ ID NO: 3 in transmembrane transport) to a molecule of SEQ ID NO: 3 or 4.

[0199] As used herein, the term "non-conservative" refers to substituting an amino acid residue for a different amino acid residue that has different chemical properties. Non-limiting examples of non- conservative substitutions include aspartic acid (D) being replaced with glycine (G); asparagine (N) being replaced with lysine (K); and alanine (A) being replaced with arginine (R).

[0200] The term "derivative" as used herein refers to peptides which have been chemically modified, for example by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or addition of other molecules. A molecule is also a "derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half life, etc. The moieties can alternatively decrease the toxicity of the molecule, or eliminate or attenuate an undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, PA (1990).

[0201] The term "functional" when used in conjunction with "derivative" or "variant" refers to a protein molecule which possesses a biological activity that is substantially similar to a biological activity of the entity or molecule of which it is a derivative or variant. By "substantially similar" in this context is meant that the biological activity, e.g., transmembrane transport of associated polypeptides is at least 50% as active as a reference, e.g., a corresponding wild-type polypeptide, and preferably at least 60% as active, 70% as active, 80% as active, 90% as active, 95% as active, 100% as active or even higher (i.e., the variant or derivative has greater activity than the wild-type), e.g., 110% as active, 120% as active, or more.

[0202] The term "recombinant" as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term recombinant as used with respect to a protein or polypeptide, means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0203] The term "vectors" refers to a nucleic acid molecule capable of transporting or mediating expression of a heterologous nucleic acid to which it has been linked to a host cell; a plasmid is a species of the genus encompassed by the term "vector." The term "vector" typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and/or maintenance in a host cell. Vectors capable of directing the expression of genes and/or nucleic acid sequence to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility are often in the form of "plasmids" which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome, and typically comprise entities for stable or transient expression or the encoded DNA. Other expression vectors that can be used in the methods as disclosed herein include, but are not limited to plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages or viral vectors, and such vectors can integrate into the host's genome or replicate autonomously in the particular cell. A vector can be a DNA or RNA vector. Other forms of expression vectors known by those skilled in the art which serve the equivalent functions can also be used, for example self replicating extrachromosomal vectors or vectors which integrates into a host genome. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.

Methods of measuring or detecting protein-protein interaction

[0204] Methods of measuring or detecting protein-protein interaction are well known. One skilled in the art can determine PA binding activity, for example, by mixing and incubating PA63 with LFn for a period of time, chemically cross-linking any complex formed and analysis of the covalently linked complex by gel electrophoresis or by radioactivity counting as described by Quinn CP. et. al., 1991, J. Biol. Chem. 266:20124-20130. Briefly, the binding assay is determined at 5°C by competition with radiolabeled 125 I-LFn. Native LF or full-length N-terminal (amino acid 1-288) LFn is radiolabeled (-7.3 x 106 cpm/μg protein) using Bolton-Hunter reagent (Amersham Corp). For binding studies, J774A.1 cells cultured in 24-well tissue culture plates are cooled by incubating at 4°C for 60 min and then placing the plates on ice. The medium is then replaced with cold (4°C) minimal essential medium containing Hanks' salts (GIBCO^® /BRL) supplemented with 1% (w/v) bovine serum albumin and 25 mM HEPES (binding medium). Native PA (0.1 g/ml) is added with radiolabeled native LF (¹²⁵I-LF, 0.1 μg/ml, 7.3 x 106 cpm/μg) and the plates incubated for 14 h on wet ice. Mutant LF proteins were assayed at varying concentrations for their ability to compete with native ¹²⁵I-LF. For quantitation of bound, radiolabeled LF, cells were gently washed twice in cold binding medium, once in cold Hanks' balanced salt solution, solubilized in 0.50 ml of 0.1 M NaOH, and counted in a gamma counter (Beckman Gamma 9000).

Methods of determining membrane translocation

[0205] Methods of determining membrane translocation are well known in the art, for example, in Wesche, J., et. al., 1998, Biochemistry 37: 15737-15746 and Sellman, B. R.,et. al, 2001, J. Biol. Chem. 276: 8371-8376. For example, CHO-Kl cells in a 24-well plate are chilled on ice, washed, and incubated on ice for 2 h with any of the LFn-target antigen fusion polypeptides described herein (or a conservative substitution variant thereof or fragments of domain I) that have been labeled with [³⁵S] methionine in an in vitro transcription/translation system (Promega). The cells then are washed with ice-cold PBS at pH 5.0 or 8.0, incubated at 37°C for 1 min, and either treated with Pronase to digest residual untranslocated ³⁵S at the cell surface or left untreated as controls. The cells are then lysed, and ³⁵S liberated into the lysis buffer is assayed. The percent translocation is defined as decay per minute (dpm) protected from Pronase/dpm bound to cells x 100. The cell lysate of cells incubated with fusion polypeptides or fragments of domain I that facilitate transmembrane delivery would have higher percent translocation. [0206] Alternatively, green fluorescent protein fused to LFn, LF or smaller fragments of domain I (e. g. LFn-GFP) can be used to assay for membrane translocation capability, as described in N. Kushner, et. al., 2003, Proc. Natl Acad Sci U S A. 100:6652-6657. Briefly, HeLa cells (American Type Culture Collection) are grown on collagen-treated chamber slides (BD Science) to reach -80% confluence and incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. After washing, GFP fluorescence is compared between GFP and GFP-LFn treated samples. Membrane translocation is evidenced by GFP signal greater in the LFn-GFP-treated cells than in cells treated with GFP alone. Some incubations can also be performed in the presence of 100 μg/ml Texas red-conjugated transferrin (INVITROGEN™ Inc., Molecular Probes) as a marker for the endocytic pathway. For the transferrin experiments, cells are washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS. For antibody labeling, slides are then incubated on ice for 15 min in 50 mM NH₄Cl in PBS and then in PBS containing 0.1% saponin for 20 min on ice. After further washing in PBS, slides are incubated at room temperature for 1 hr in a moisture chamber with PBS containing 4% donkey serum and the following primary antibodies: mouse anti-early endosome antigen 1 (EEA-I) (BD Laboratory) to stain early endosomes, mouse anti-Lamp 1 and anti-Lamp2 (Developmental Studies Hybridoma Banks, University of Iowa, Iowa City) to stain late endosomes and lysosome, mouse Ab-I (Oncogene) to stain the Golgi apparatus, mouse anti-mitochondrial antibody from CALBIOCHEM^®, and rabbit anti-calreticulin (STRESSGEN^® Biotechnologies, Victoria, Canada). Cells are then processed for secondary antibody staining and microscopy. Fusion LFn-GFP that promotes transmembrane delivery would be visualized in the interior of the cell. The antibody markers will further indicate sub-cellular localization of the translocated GFP.

Zinc metalloproteinase activity by FRET analysis

[0207] Assays of LF peptidolytic activity based on cleavage of the FRET-quenched substrate MAPKKide can be carried out according to a modification of the method of Cummings et al. (2002, Proc. Natl. Acad. Sci. USA 99:6603-6606.). MAPKKide (o-aminobenzoyl [o-ABZ]/2,4-dinitrophenyl [DNP]), a synthetic peptide containing the o-ABZ donor and DNP acceptor groups separated by a cleavage site specific for anthrax LF, was purchased from List Biological Labs. Digestion of MAPKKide by LF was carried out in Dulbecco's phosphate -buffered saline (DPBS) (HyClone, Logan Utah), pH 8.2, as recommended by the manufacturer and was followed in a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) or in a, LS-5 fluorescence spectrophotometer (Perkin-Elmer, Wellesley, MA) using a λ excitation (ex) value of 320 nm and a λ emission (em) value of 420 nm. LF was preincubated with indicated concentrations of putative inhibitors for 10 min at room temperature, and the reaction was initiated by addition of indicated concentrations of the substrate to a 100-μl or 500- μl reaction mixture.

Antigenic polypeptides from the influenza viruses

[0208] Influenza is a major cause of respiratory illness in adults and children worldwide. The three species that infect humans are Influenza virus A, Influenza virus B, and Influenza virus C. These are RNA viruses of the family Orthomyxoviridae . The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The influenza A virus can be subdivided into different serotypes based on the antibody response to the hemagglutinin (HA), and neuraminidase (NA) glycoproteins on the outside of the viral particles, forming the basis of the H and N distinctions in, for example, H5N1. Influenza B almost exclusively infects humans and is less common than influenza A. Influenza C virus infects humans and pigs and can cause severe illness and local epidemics. However, influenza C is less common than the other types and usually seems to cause mild disease in children.

[0209] Influenza viruses A, B and C are very similar in structure. The virus particle is 80-120 nanometers in diameter and usually roughly spherical, although filamentous forms can occur. The genome of these viruses is not in a single piece of nucleic acid; instead, it contains seven or eight pieces of segmented negative-sense RNA. Both influenza A and B viruses have a segmented genome consisting of eight negative-stranded RNA segments, six of which code for the internal proteins nucleoprotein (NP), matrix proteins (Ml, M2), nonstructural proteins (NSl and NS2), and RNA polymerase proteins (PA, PBl, PB 2), and two of which code for the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA and NA are large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Viral infection is initiated by HA binding to the sialic acid-containing host cell receptor, while NA cleaves the sialic acid to promote the release of virus from infected cells. Functional coordination of these two proteins is required for efficient virus replication. As such, the HA and NA proteins are the common targets for antiviral drugs. In addition, they are the favored target antigens to which antibodies can be raised and the favored target antigens in vaccine compositions for immunization purposes. Influenza virus C has 7 RNA segments and encodes 9 proteins.

[0210] Hemagglutinin A (HA) is encoded on segment 4 of the segmented RNA genome. HA is involved in viral attachment to terminal sialic acid residues on host cell glycoproteins and glycolipids. After viral entry into an acidic endosomal compartment of the cell, HA is also involved in fusion with the cell membrane, which results in the intracellular release of the virion contents. HA is synthesized as an HAO precursor that forms noncovalently bound homotrimers on the viral surface. The HAO precursor is cleaved by host proteases at a conserved arginine residue to create two subunits, HAl and HA2, which are associated by a single disulfide bond This cleavage event is required for productive infection. [0211] HA is a critical determinant of the pathogenicity of avian influenza viruses, with a clear link between HA cleavability and virulence. The HA proteins of highly pathogenic H5 and H7 viruses contain multiple basic amino acid residues at the cleavage sites which are recognized by ubiquitous proteases, furin and PC6. For this reason, these viruses can cause systemic infections in poultry. Two groups of proteases are responsible for HA cleavage. The first group recognizes a single arginine and cleaves all HAs. Members of this group include plasmin, blood-clotting factor X-like proteases, tryptase Clara, miniplasmin, and bacterial proteases. The second group of proteases that cleaves HA proteins comprises the ubiquitous intracellular subtilisin-related endoproteases furin and PC6. These enzymes are calcium dependent, have an acidic pH optimum, and are located in the Golgi and/or trans-GoIgi network. [0212] The mature HA forms homotrimers. The crystallographic study of HA revealed the major features of the trimer structure: (a) a long fibrous stem that is comprised of a triple- stranded coiled coil of α-helices derived from the three HA2 parts of the molecule, and (b) the globular head, which is also comprised of three identical domains whose sequences are derived from the HAl portions of the three monomers.

[0213] Influenza A virus RNA segment 5 encodes NP, a polypeptide of 498 amino acids in length, which is rich in arginine, glycine and serine residues and has a net positive charge at neutral pH. The majority of the polypeptide has a preponderance of basic amino acids and an overall predicted pi of 9.3, but the C-terminal 30 residues of NP are, with a pi of 3.7, markedly acidic. In influenza B and C viruses, the length of the homologous NP polypeptide is 560 and 565 residues, respectively. Alignment of the predicted amino acid sequences of the NP genes of the three influenza virus types reveals significant similarity among the three proteins, with the type A and B NPs showing the highest degree of conservation. Phylogenetic analysis of virus strains isolated from different hosts reveals that the NP gene is relatively well conserved, with a maximum amino acid difference of less than 11 % (See, Shu et al., 1991, J. Virology 67:2723-2729).

[0214] The major viral protein in the ribonucleoprotein (RNP) complex of an influenza virus is the NP. It is a structural protein than encapsulates the RNA. It is believed to be a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. Both NP and Ml are required in packaging the ribonucleoprotein complexes, NP coats and protects the sugar phosphate backbone of the viral RNA. Ml is critical for export of the complex from the nucleus, mediating the interaction of the RNP complex with the viral NEP/NS2 protein, which in turn interacts with host cell CRMl nuclear-export protein.

[0215] The matrix proteins (Ml, M2) are found on segment 7 of the virus genome. Ml protein is encoded by an mRNA that is colinear, while M2 protein is synthesized from spliced mRNA. The Ml protein is the most abundant component of virions and forms a membrane matrix between ribonucleoprotein (RNP) cores and lipid envelope. Ml plays a role in turning off RNA replication. After Ml binding, RNP cores become exportable from nuclei into the cytoplasm Thus, Ml has a regulatory role in switching from RNA replication to virus maturation.

[0216] M2 protein is a transmembrane protein composed of three Domains: 1) 24 residues representing the N-terminal region, 2) 19 hydro-phobic residues that serve as a membrane anchor, and 3) 54 residues near the C-terminal in the cytoplasmic domain. The M2 protein has been found to play a role in Influenza replication and assembly of virion particles. M2 protein is an acid-activated ion channel for virus replication. When the environmental pH is lower than a threshold, the M2 channel is activated and selectively transports protons across the membrane from the extracellular side to the cytoplasmic side. It is crucial for the uncoating process of the RNP complex in the endosome. When the virion is internalized into the endosome the M2 channel acidifies the virion interior, promoting the dissociation of the viral matrix protein (Ml) from the ribonucleoprotein (RNP), thus allowing the transport of the RNP from the virion into the cell's nucleus. For some influenza virus subtypes, the M2 channel can also elevate the intravesicular pH of the trans Golgi network, preventing the viral protein haemagglutinin, which is transported to the cell surface through the trans Golgi network, from incorrect maturation in an otherwise low pH environment.

[0217] Influenza A virus RNA segment 8 encodes the non-structural protein 1 and 2 (NSl and 2). NSl is a homodimeric RNA-binding protein that is required for viral replication. NSl binds polyA tails of mRNA keeping them in the nucleus. NSl inhibits pre-mRNA splicing by tightly binding to a specific stem-bulge of U6 snRNA (Nat. Struct. Biol. 1997, 4:891-895).

[0218] Neuraminidase (NA) is encoded on segment 6. NA cleaves terminal sialic acid residues of influenza A cellular receptors and is involved in the release and spread of mature virions. It may also contribute to initial viral entry. NA is the target of inhibitor drugs such as oseltamivir and zanamivir. [0219] Influenza RNA polymerase is composed of three subunits: PB 1 , PB2 and PA. This RNA- dependent RNA polymerase is responsible for replication and transcription of virus segments. It binds the promoter sequence of the encapsidated viral RNA, displays an endonuclease activity involved in cap- stealing, and cleaves cellular pre-mRNA to generate primers for viral transcription.

Lethal factor (LF) of Bacillus anthracis and the N -terminal fragment (LFn)

[0220] B. anthracis is the causative agent of anthrax in animals and humans. The toxin produced by B. anthracis consists of two bipartite protein exotoxins, lethal toxin (LT) and edema toxin. LT is composed of protective antigen (PA) and lethal factor (LF), whereas edema toxin consists of PA and edema factor (EF). None of these three components, PA, LF, and EF, alone is toxic. Once combined however, edema toxin causes edema and LT causes death by systemic shock in animals and humans. Consistent with its critical role in forming both toxins, PA has been identified as the protective component in vaccines against anthrax. The molecular mechanism of anthrax toxin action is currently hypothesized as follows: PA is a 735-amino acid polypeptide, a multi-functional protein that binds to cell surface receptors, mediates the assembly and internalization of the complexes, and delivers them to the host cell endosome. Once PA is attached to the host receptor, it must then be cleaved by a host cell surface (furin family) protease before it is able to bind LF. The cleavage of the N-terminus of PA enables the C-terminal fragment to self-associate into a ring-shaped heptameric complex (prepore) that can bind LF or EF competitively. The cleaved PA is a 63-kDa molecule (PA63) capable of forming a ring-shaped heptamer in the plasma membrane of the targeted cell (Milne et al., (1994) J. Biol. Chem. 269, 20607-20612, Petosa, et al., (1997) Nature (London) 385, 833-838). The PA heptamer then binds either EF or LF, which are internalized by endocytosis into intracellular compartments called endosomes. Natural cellular processes lead to acidification of the endosome, which triggers a conformational change in PA63, causing it to insert into the endosomal membrane and translocates the toxic enzymes into the host cell interior (cytosol), presumably by means of a pore formed by the heptamer. Once inside the cytosol, LF and EF do their damage to the host cell defense system. LF is a metalloproteinase that cleaves six members of the MAPKK family (Vitale G, . et. al., 2000, Biochem. J. 352, 739-745) of intracellular signaling proteins, removing the specific fragment from individual MAPKKs that are crucial for immediate interaction with other signaling proteins. This action by LF rapidly blocks the signals that would normally recruit other immune cells to fight the infection (Duesbury NS,. et. al., 1998, Science 280, 734-737). EF, on the other hand, is a calmodulin-activated adenylyl cyclase that increases the concentration of a messenger molecule (cAMP) needed for regulated cell functions (Leppla SH., et. al., 1982, Proc. Natl. Acad. Sci. USA 79, 3162-3166) to abnormal levels, causing accumulation of fluids within and between cells, and hence edema. The disruption of normal signaling pathways result in cell lysis, the sudden release of messenger molecules, and toxic shock.

[0221] Anthrax lethal factor or LF is a protein, encoded by GenBank Accession Number M29081 (Gene ID No: 143143), that is naturally produced by B. anthracis and that has MAPKK protease activity. The gene encoded B. anthracis LF is a 809 amino acid polypeptide while the mature B. anthracis LF is a 796 amino acid polypeptide after cleavage of the N-terminal leader peptide. Deletion analysis of LF shows that the PA binding domain is located within the amino-terminus of LFn. Mutational studies demonstrate the PA binding domain is located within the region of amino acids 34 to 288 of the LF polypeptide of SEQ ID NO: 1, and within the region of amino acids 1 to 254 of the LF polypeptide of SEQ ID NO: 2 (Arora et al., J. Biol. Chem. 268:3334 3341 (1993); Milne, et al., (1995) MoI. Microbiol. 15, 661-66). The three-dimensional atomic resolution structures of LF have now been solved by X-ray crystallography. Pannifer et al., describe the crystal structure of LF and its complex with a 16-amino acid residue (16-mer) peptide representing the N-terminus of its natural substrate, MAPKK-2, in Nature vol. 414, pg. 229-233 (2001) as a protein that comprises four structural domains: domain I binds the membrane -translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain I is perched on top of the other three domains, which are intimately connected and comprise a single folding unit. The only contacts between domain I and the rest of the molecule are with domain II, and these chiefly involve charged polar and water-mediated interactions. The nature of the interface is consistent with the ability of a recombinant N-terminal fragment (residues 1-254, excluding the signal peptide) to be expressed as a soluble folded domain that maintains the ability to bind PA and enables the translocation of heterologous fusion proteins into the cytosol (Ballard, J. D., et. al., 1996, Proc. Natl Acad. Sci. USA 93, 12531-12534; Goletz, T. J. et al., 1997, Proc. Natl Acad. Sci. USA 94, 12059-12064). Moreover, deletion of the first 36 residues of LFn had no effect on its binding to PA or LF ability to be translocated across membranes (D. Borden Lacy, et.al., 2002, J. Biol. Chem., 277:3006- 3010). Domain I consists of a 12-helix bundle that packs against one face of a mixed four-stranded β- sheet, with a large (30-residue) ordered loop, Ll, between the second and third -strands forming a flap over the distal face of the sheet (see Fig. 1). The exact docking site on domain I for PA is unknown, but the integrity of the folded domain seems to be required, because a series of insertion and point mutants of buried residues in domain I that presumably disrupt the fold abrogate binding of PA and toxicity (Quinn, C. P., et. al., 1991, J. Biol. Chem., 266: 20124-20130; Gupta, P., et. al., 2001, Biochem. Biophys. Res. Comm., 280:158-163). In addition, LFn has been shown to deliver exogenous protein antigens to the major histocompatibility complex class I pathway in the cytosol of B-cells, CTL-cells and macrophages in the absence of PA (Huyen Cao, et. al., 2002, The Journal of Infectious Diseases; 185:244-251; N. Kushner, et. al., 2003, Proc Natl Acad Sci U S A. 100: 6652-6657). The PA-independent LFn delivery of LFn-fusion proteins depends on functional transport-associated proteins for intracellular antigen processing and transport into the endoplasmic reticulum for binding to MHC class I molecules. [0222] An abrupt turn at the end of the last helix of domain I leads directly into the first helix of domain II (residues 263-297 and 385-550). Although sequence-based comparisons failed to yield any homology, the structural similarity with the catalytic domain of the B. cereus toxin, VIP2 (Protein Data Bank accession code 1QS2), is outstanding. Domain II and VIP2 superimpose with an RMSD of 3.3 A and a sequence identity of 15%, as determined by DALI (Holm, L. & Sander, 1997, Nucleic Acids Res. 25, 231-234). VIP2 contains an NAD-binding pocket and conserved residues involved in NAD binding and catalysis. Domain II lacks these conserved residues; moreover, a critical glutamic acid that is conserved throughout the family of ADP ribosylating toxins (Carroll, S. F. & Collier, R. J., 1984, Proc. Natl Acad. Sci. USA 81, 3307-3311) is replaced by a lysine (K518). It is therefore expected that domain II does not have ADP-ribosylating activity.

[0223] Domain III is a small α-helical bundle with a hydrophobic core (residues 303-382), inserted at a turn between the second and third helices of domain II. Sequence analysis has revealed the presence of a 101-residue segment comprising five tandem repeats (residues 282-382), and suggested that repeats 2-5 arose from a duplication of repeat 1. The crystal structure reveals that repeat 1 actually forms the second helix-turn element of domain II, whereas repeats 2-5 form the four helix-turn elements of the helical bundle, suggesting a mechanism of creating a new protein domain by the repeated replication of a short segment of the parent domain. Domain III is required for LF activity, because insertion mutagenesis and point mutations of buried residues in this domain abrogate function (Quinn, C. P., et. al., 1991, J. Biol. Chem. 266, 20124-20130). It makes limited contact with domain II, but shares a hydrophobic surface with domain IV. Its location is such that it severely restricts access to the active site by potential substrates such as the loops of a globular protein; that is, it contributes towards specificity for a flexible 'tail' of a protein substrate. It also contributes sequence specificity by making specific interactions with the substrate (see below).

[0224] Domain IV (residues 552-776) consists of a nine-helix bundle packed against a four-stranded - sheet. Sequence comparisons had failed to detect any homology with other proteins of known structure beyond the HExxH motif. The three-dimensional structure reveals that the β-sheet and the first six helices can be superimposed with those of the metalloprotease thermolysin, with an RMSD of 4.9 A over 131 residues. Large insertions and deletions occur elsewhere within the loops connecting these elements, so that the overall shapes of the domains are quite different. In particular, a large ordered loop (L2) inserted between strands 42 and 43 of the sheet partly obscures the active site, packs against domain II, and provides a buttress for domain III.

[0225] A zinc ion (Zn2+) is coordinated tetrahedrally by a water molecule and three protein side chains, in an arrangement typical of the thermolysin family. Two coordinating residues are the histidines from the HExxH motif (His 686 and His 690) lying on one helix (44), as expected. The structure reveals that the third coordinating residue is GIu 735 from helix 46. GIu 687 from the HExxH motif lies 3.5 A from the water molecule, well positioned to act as a general base to activate the zinc-bound water during catalysis. The hydroxyl group of a tyrosine residue (Tyr 728) forms a strong hydrogen bond (0-0 distance 2.6 A) to the water molecule, on the opposite side of GIu 687, and probably functions as a general acid to protonate the amine leaving group.

[0226] The gene encoded 809 amino acid polypeptide B. anthracis LF has seven potential N- glycosylation sites located at asparagine positions 62, 212, 286, 478, 712 736, and 757. Within the LFn (1-288), there are three potential N-glycosylation sites, at asparagine positions 62, 212, and 286, all of which have potential of > 0.51 according to the NetNGlyc 1.0 Prediction software from the Technical University of Denmark. The NetNglyc server predicts N-Glycosylation sites in proteins using artificial neural networks that examine the sequence context of Asn-Xaa-Ser/Thr sequons. [0227] The gene encoded 809-aa polypeptide B. anthracis LF is not predicted to have any O- glycosylation sites according to the NetOGlyc 3.1 Prediction software from the Technical University of Denmark. The NetOglyc server produces neural network predictions of mucin type GaINAc O- glycosylation sites in proteins.

[0228] "LFn polypeptides" include LF polypeptide fragments represented by SEQ ID Nos. 3 and 4, as well as recombinant LFn, and functional LFn, fragments and variants that retain the function to deliver an LFn-fused target antigen polypeptide to the cytosol of an intact cell, preferably a living cell. The term "LFn polypeptide" therefore includes functional LFn homologues such as polymorphic variants, alleles, mutants, and closely related interspecies variants that have at least about 60% amino acid sequence identity to LFn and have the function to deliver a fused polypeptide target antigen to the cytosol of a cell, as determined using the assays described herein. In particular embodiments, the LFn polypeptides are substantially identical to LFn of SEQ ID NO: 3 and SEQ ID NO: 4 as disclosed herein. In other embodiments, the LFn polypeptides are conservative substitution mutants of LFn of SEQ ID NO: 3 and SEQ ID NO: 4 as disclosed herein. These conservative substitution mutants of LFn can also function to deliver a fused polypeptide target antigen to the cytosol of a cell, as determined using the assays described herein. In some embodiments, some functional polymorphic variants, alleles, mutants, and closely related interspecies variants of LFn that function to deliver a target antigen polypeptide to an intact cell can be determined by the methods and assays as disclosed in U.S. Patent Application 10/473,190 which is incorporated herein by reference.

[0229] The inventors have discovered that a fragment of LFn which is about 250 amino acids or less, or about 150 amino acids or less, or about 104 amino acids or less, is able to deliver the fused target antigen NP to a cell and is useful in the methods and compositions described herein.

[0230] In one embodiment, an LFn polypeptide as described herein comprises a non-functional binding site for PA, and thus is a mutant of LFn which does not result in functional binding with PA. Such mutants include, but are not limited to mutants altered at one or more of the residues critical for interacting with PA, such as a mutation in one or more of the following residues: Y22; Ll 88; Dl 87; Y226; L235; H229 (see Lacy et al., J. Biol. Chem., 2002; 277; 3006-3010); D106A; Y108K; E135K; D136K; N140A and K143A (see Melnyk et al., J. Biol. Chem., 2006; 281; 1630-1635 and Cunningham et al., PNAS, 2002; 99; 70497052, which are incorporated herein in their entirety by reference).

Production of polypeptide using a baculovirus system

[0231] In one embodiment, any of the polypeptides described herein is produced by expression from a recombinant baculovirus vector. In another embodiment, any of the polypeptides described herein is expressed by an insect cell. In yet another embodiment, any of the polypeptides described herein is isolated from an insect cell. There are several benefits of protein expression with baculovirus in insect cells, including high expression levels, ease of scale-up, production of proteins with posttranslational modifications, and simplified cell growth. Insect cells do not require CO2 for growth and can be readily adapted to high-density suspension culture for large-scale expression. Many of the post-translational modification pathways present in mammalian systems are also utilized in insect cells, allowing the production of recombinant protein that is antigenically, immunogenically, and functionally similar to the native mammalian protein.

[0232] Baculoviruses are DNA viruses in the family Baculoviridae. These viruses are known to have a narrow host-range that is limited primarily to Lepidopteran species of insects (butterflies and moths). The baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV), which has become the prototype baculovirus, replicates efficiently in susceptible cultured insect cells. AcNPV has a double- stranded closed circular DNA genome of about 130,000 base -pairs and is well characterized with regard to host range, molecular biology, and genetics.

[0233] Many baculoviruses, including AcNPV, form large protein crystalline occlusions within the nucleus of infected cells. A single polypeptide, referred to as a polyhedrin, accounts for approximately 95% of the protein mass of these occlusion bodies. The gene for polyhedrin is present as a single copy in the AcNPV viral genome. Because the polyhedrin gene is not essential for virus replication in cultured cells, it can be readily modified to express foreign genes. The foreign gene sequence is inserted into the AcNPV gene just 3' to the polyhedrin promoter sequence such that it is under the transcriptional control of the polyhedrin promoter.

[0234] The Baculovirus Expression Vector System (BEVS) is a safe and rapid method for the abundant production of recombinant proteins in insect cells and insects pioneered in the laboratory of Dr. Max D. Summers.

[0235] Baculovirus expression systems are powerful and versatile systems for high-level, recombinant protein expression in insect cells. Expression levels up to 500 mg/1 have been reported using the baculovirus expression system, making it an ideal system for high-level expression. Recombinant baculoviruses that express foreign genes are constructed by way of homologous recombination between baculovirus DNA and chimeric plasmids containing the gene sequence of interest. Recombinant viruses can be detected by virtue of their distinct plaque morphology and plaque -purified to homogeneity. [0236] Baculoviruses are particularly well-suited for use as eukaryotic cloning and expression vectors. They are generally safe by virtue of their narrow host range which is restricted to arthropods. The U.S. Environmental Protection Agency (EPA) has approved the use of three baculovirus species for the control of insect pests. AcNPV has been applied to crops for many years under EPA Experimental Use Permits.

[0237] AcNPV wild type and recombinant viruses replicate in a variety of insect cells, including continuous cell lines derived from the fall armyworm, Spodoptera frugiperda (Lepidoptera; Noctuidae). S. frugiperda cells have a population doubling time of 18 to 24 hours and can be propagated in monolayer or in free suspension cultures.

[0238] Recombinant fusion proteins described herein can be produced in insect cells including, but not limited to, cells derived from the Lepidopteran species S. frugiperda. Other insect cells that can be infected by baculovirus, such as those from the species Bombyx mori, Galleria mellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as a suitable substrate to produce recombinant proteins described herein.

[0239] Baculovirus expression of recombinant proteins is well known in the art and is described in U.

S. Patent Nos. 4,745,051, 4,879,236, 5,179,007, 5,516,657, 5,571,709 and 5,759,809 which are all incorporated by reference in their entirety. It will be understood by those skilled in the art that the expression system is not limited to a baculovirus expression system. What is important is that the expression system directs the N-glycosylation of expressed recombinant proteins. The recombinant proteins described herein can also be expressed in other expression systems such as Entomopox viruses

(the poxviruses of insects), cytoplasmic polyhedrosis viruses (CPV), and transformation of insect cells with the recombinant gene or genes constitutive expression.

[0240] The most common expression vector system is from the insect baculovirus A. californica nuclear polyhedrosis virus (AcNPV). AcNPV has a genome of ca. 130 kilobases (kb) of double-stranded, circular DNA and it is the most extensively studied baculovirus. Miller, L.K., J Virol. 1981, 39:973-976.

AcNPV has a biphasic replication cycle and produces a different form of infectious virus during each phase. Between 10 and 24 h postinfection (p.i.), extracellular virus is produced by the budding of nucleocapsids through the cytoplasmic membrane. By 15 to 18 h p.i., nucleocapsids are enveloped within the nucleus and embedded in a paracrystalline protein matrix, which is formed from a single major protein called polyhedrin. In infected S. frugiperda (fall armyworm, Lepidoptera, Noctuidae) cells,

AcNPV polyhedrin accumulates to high levels and constitutes 25% or more of the total protein mass in the cell; it may be synthesized in greater abundance than any other protein in a virus-infected eukaryotic cell.

[0241] In one embodiment, any of the polypeptides described herein is produced using a Baculovirus

Expression Vector System (BEVS), by infecting lepidopteran insect cells with a recombinant baculovirus vector comprising a polynucleotide encoding the polypeptide and culturing the insect cells to produce the polypeptide.

[0242] In some embodiments, the Baculovirus Expression Vector System (BEVS) uses lepidopteran insect S. frugiperda cells.

[0243] The gene encoding LF has been cloned and sequenced, and has been assigned Genbank accession no. M29081 (Robertson and Leppla, 1986, Gene 44:71 78; Bragg and Robertson, 1989, Gene

81:45 54; see also U. S. Pat. Nos. 5,591,631 and 5,677,274; see generally Leppla, Anthrax Toxins, in

Bacterial Toxins and Virulence Factors in Disease (Handbook of natural toxins, Vol. 8., Moss et al., eds.,

1995).

[0244] The gene encoding Ml of the Influenza A virus (A/chicken/Chile/4977/02(H7N3)), has been assigned Genbank Accession No. AY303656 (Gene ID: GI: 34597766). The amino acid sequence

(Genbank Accession No. AAQ77440.1 (Gene ID: GI: 34597767)). The making of a recombinant Ml protein are described in C. Elster, et. al., 1997 (J. Gen. Virol, 78:1589-1596) and Baylor, NW et. al.,

1988 (Virology, 163:618-621). These references are incorporated hereby reference in their entirety. [0245] The gene encoding NP of the Influenza A virus (A/Paris/908/97(H3N2)) has been assigned Genbank Accession No. AF483604 (Gene ID: GL21902317). The making of a recombinant NP protein are described in B. Lin and C. Lai, 1983, (J. Virol., 45:434-438), in Harmon et al., 1989, (J. Med. Virol. 24:25-30) and ( in FAN Hong-bo et. al., 2007 (Chinese virology, Vol.22 No. 01, ISSN: 1003-5125 (2007) 01-0046-07). These references are incorporated hereby reference in their entirety. [0246] The gene encoding M2 of the Influenza A virus (A/chicken/Chile/4977/02(H7N3)) has been assigned Genbank Accession No. AY303656 (Gene ID: GI: 34597766). The amino acid sequence (Genbank Accession No. AAQ77441.1 (Gene ID: GI: 34597768)). Expression of an influenza a M2 protein in baculovirus is described in U. S. Patent No. 5,290,686 which is incorporated hereby reference in its entirety.

[0247] The coding DNA sequences are typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression. These intermediate vectors are typically cloning plasmids, (e. g. pPUC19, PBLUESCRIPT^®-SK) or shuttle vectors that can be propagated in a number of different hosts and to allow more efficient manipulation of DNA (e. g. the pRS YCp and pRS Yip vectors can shuttle between bacteria and Saccharomyces cerevisiae). [0248] To generate influenza virus sequences for expression in a baculovirus system, for example, Virion RNA can be extracted from gradient purified influenza B/Ann Arbor/1/86 and A/ Ann Arbor/6/60 (wild-type) viruses by standard methods (Cox et al., 1983, Bulletin of the World Health Organization 61, 143-152). cDNA copies of total viral RNA are prepared by the method of Lapeyre and Amairic (Lapeyre et al., 1985, Gene 37, 215-220) except that first-strand synthesis by reverse transcriptase was primed by using universal influenza type A or B primers complementary to the 3' untranslated region of virion RNA. The double-stranded cDNA fragment corresponding to influenza genomic RNA segment 5 and 7 (influenza A: 1565 base pairs; influenza B: 1811 base pairs) are isolated from agarose gels, purified, and ligated into the Sma I site of plasmid pUC 8 using standard methods (Maniatis et al., 2001, 3^rd edition, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.) Bacterial colonies (E. coli, HBlOl) containing recombinant plasmids with NP, Ml, or M2 inserts are identified by in situ hybridization (Maniatis et al., (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) to ³² P-labeled, oligonucleotide primers with sequences specific for influenza A or B NP, Ml or M2 genes.

[0249] LF and LFn coding sequences are described above and can be cloned by one of skill in the art or obtained from existing clones available in the art.

[0250] The first step in the production of recombinant proteins from a BEVS is the construction of a recombinant baculovirus vector, either by homologous recombination or by site specific transposition. To obtain a recombinant baculovirus vector by homologous recombination, a baculovirus transfer vector is needed. A baculovirus transfer vector is a temporary vector whose sole purpose is to enable the insertion of foreign coding DNA, under an appropriate gene promoter, into the baculovirus genome at a site that would not affect normal viral replication. The baculovirus transfer vector comprises a portion of the baculovirus genomic sequence that spans the intended insertion site of the foreign coding DNA. The most common regions contain the polyhedrin or plO gene. Both are dispensible for viral replication in cell culture and insect larvae, and the production of infectious extracellular virus. Both proteins are highly expressed at the very late phase of viral replication and effect high level of transcription of the foreign gene when inserted back into the viral genome. A typical baculovirus transfer vector comprises a promoter, a transcriptional terminator, and most often native viral sequences and regions flanking both sides of the promoter that are homologous to the target genes in the viral genome. The region between the promoter and the transcriptional terminator can have multiple restriction enzyme digestion sites for facilitating cloning of the foreign coding sequence, in this instance, the coding DNA sequence for an LF polypeptide, e.g., an LFn polypeptide and an influenza target antigen. Additional sequences can be included, e.g., signal peptides and/or tag coding sequences,such as His-tag, MAT-Tag, FLAG tag, recognition sequence for enterokinase, honeybee melittin secretion signal, beta-galactosidase, glutathione S-transf erase (GST) tag upstream of the MCS for facilitating the secretion, identification, proper insertion, positive selection of recombinant virus, and/or purification of the recombinant protein. Subsequent to the construction of the baculovirus transfer vector, it is mixed with AcNPV viral DNA and co-transfected into insect cells to establish an infection. The native polyhedrin gene is removed by a double-cross over homologous recombination event and replaced by the foreign coding sequence to be expressed in the insect cells. Inactivation of the polyhedrin gene by deletion or by insertion results in mutants that do not produce occlusions in infected cells. These occlusion-negative viruses form plaques that are different from plaques produced by wild-type viruses, and this distinctive plaque morphology is useful as a means to screen for recombinant viruses.

[0251] A good number of baculovirus transfer vectors and the corresponding appropriately modified host cells are commercially available, for example, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 from BD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from NOVAGEN^®, and pPolh-FLAG and pPolh-MAT from SIGMA ALDRICH^®. One skilled in the art would be able to clone and ligate the coding region of the B. anthracis lethal factor N-terminal (LFn) portion with the coding region of a target antigen polypeptide or fragment thereof to construct a chimeric coding sequence for a fusion polypeptide comprising LFn and the target antigen polypeptide or fragment thereof using specially designed oligonucleotide probes and polymerase chain reaction (PCR) methodologies that are well known in the art. One skilled in the art would also be able to clone and ligate the chimeric coding sequence for a fusion protein into a selected baculovirus transfer vector. The coding sequences of LFn and the target antigen polypeptide or fragment thereof should be ligated in-frame and the chimeric coding sequence should be ligated downstream of the promoter, and between the promoter and the transcription terminator. Subsequent to that, the recombinant baculovirus transfer vector is transfected into regular cloning Escherichia coli, such as XLl Blue. Recombinant E. coli harboring the transfer vector DNA is then selected by antibiotic resistance to remove any E. coli harboring non-recombinant plasmid DNA. The selected transformant E. coli are grown and the recombinant vector DNA is subsequently purified for transfection into S. frugiperda (SF) cells. [0252] As an example, the oligonucleotide 5'-GGAGGAACATATGGCGGGCGGTCATGGTGATG-S' (SEQ. ID. No. 19) can be used to introduce an Ndel site and serve as a forward primer in the amplification of the coding DNA sequence for LFn-(amino acids 1-263) and the oligonucleotide 5'- CTAGGATCCTT ACCGTTGATCTTT AAGTTCTTCC-3' (SEQ. ID. NO. 20) can be used to introduce a BamHl site and act as the reverse primer. PCR amplification is performed using the cDNA template according to GenBank Accession No. M29081. The forward primers for LFn-(28-263), LFn-(33-263), LFn-(37-263), LFn-(40-263), and LFn-(43-263) can be designed accordingly permit the PCR amplification of the coding sequence of the appropriate truncated LFn and also introduce an Ndel site. [0253] As an example, for cloning the full-length NP, the oligonucleotide CT AGAAGTCC ATGGCGTCCCAAGGCACCAAACGG (SEQ. ID. No. 21) can be used to introduce a BamHl site and 5'- CTAGAGCTCATTGTCGTACTCTTCTGCATTGTC -3' can be used to introduce a Xhol site (SEQ. ID. No.22)

[0254] The common BamHl site at the end of the amplified coding sequence of LFn and at the beginning of the amplified coding sequence of NP facilitates the ligation of the two separate amplified coding sequences into a chimeric or fusion coding sequence. The ligation of the two separate amplified coding sequences should be such that NP is in frame with LFn and there is no translation stop codon around the ligation site. The fusion coding sequence can then be digested with Ndel and Xhol and ligated into a selected baculovirus transfer vector that has Ndel and Xhol sites with the appropriate orientation. The newly constructed baculovirus transfer vector can be transformed into E. coli DH5. E. coli transformants can be screened by digestion and verified by sequencing. After that, the baculovirus transfer vector can be isolated for co-transfection into insect cells for homologous recombination. Similar approaches can obviously be taken for cloning other influenza antigen sequences.

[0255] To obtain a recombinant baculovirus vector by site specific transposition, e. g. with Tn7 to insert foreign genes into bacmid DNA propagated in E. coli. , INVITROGEN™ Inc. provides the pFASTBAC™ plasmid and bacmid containing DH 1OB AC™ competent E. coli for constructing a recombinant baculovirus vector by site specific transposition. The coding sequence is cloned into a pFASTBAC™ plasmid and the recombinant plasmid is transformed into an DH10BAC™ competent E. coli harboring bacmid, a baculovirus shuttle vector, with a mini-attTn7 target site and a helper plasmid. The mini-attTn7 element on the pFASTBAC™ plasmid can transpose to the mini-attTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid. Colonies containing recombinant bacmids are identified by antibiotics selection and by blue/white screening, since the transposition results in the disruption of the LacZα gene that is flanked by the mini-attTn7 target site on the bacmid. The bacmid is then harvested for transfection of insect cells.

[0256] In one embodiment, a fusion polypeptide described herein has a spacer peptide, e. g., a 14- residue spacer (GSPGISGGGGGILE) (SEQ. ID. No. 23) separating the LF polypeptide (e. g., an LFn polypeptide) from the influenza polypeptide. The coding sequence of such a short spacer can be constructed by annealing a complementary pair of primers. One of skill in the art can design and synthesize oligonucleotides that will code for the selected spacer. Spacer peptides should generally have non-polar amino acid residues, such as glycine and proline.

[0257] In some instances, specific site -directed mutagenesis of the chimeric coding sequence in the baculovirus transfer vector can be performed to create specific amino acid mutations and substitutions to further promote transmembrane delivery, protein expression or protein folding. An example of an amino acid substitution include glutamate for aspartate. Site-directed mutagenesis can be carried out, e.g., using the QUIKCHANGE^® site -directed mutagenesis kit from Stratagene according to manufacture's instructions or any methods known in the art.

[0258] Standard viral DNA is used to co-transfect S. frugiperda (SF) cells. Putative recombinant viruses containing the recombinant molecules are isolated from the virus produced in these transfected monolayers. Because the polyhedrin structural gene has been removed, plaques containing the recombinant viruses can be easily identified since they lack occlusion bodies. Confirmation that these recombinants contain the desired chimeric coding sequence is established by methods well known in the art, such as hybridization with specific gene probes, plaque assays, and end point dilution.

[0259] A preferred host cell line for protein production from recombinant baculoviruses described herein is Sf900+. Another preferred host cell line for protein production from recombinant baculoviruses is Sf9. Sf900+ and Sf9 are non-transformed, non-tumorigenic continuous cell lines derived from the fall armyworm, S. frugiperda (Lepidoptera; Noctuidae).

[0260] Sf900+ and Sf9 cells are propagated at 28 ± 2°C without carbon dioxide supplementation. The culture medium used for Sf9 cells is TNMFH, a simple mixture of salts, vitamins, sugars and amino acids, supplemented with 10% fetal bovine serum. Aside from fetal bovine serum, no other animal derived products (i.e, trypsin, etc.) are used in cell propagation. Serum free culture medium (available as

Sf900 culture media, GIBCO^® BRL, Gaithersburg, Md.) can also be used to grow Sf9 cells and is preferred for propagation of Sf 900+ cells. Sf9 cells have a population doubling time of 18-24 hours and can be propagated in monolayer or in free suspension cultures. S. frugiperda cells have not been reported to support the replication of any known mammalian viruses.

[0261] Plaque assays of baculovirus transfected monolayers SF cells are well known in the art. Below is a standard protocol.

[0262] Reagents needed: Grace's Insect Medium, 2X (e.g. BD Biosciences GIBCO^® #11667), fetal bovine serum (Heat Inactivated), (e.g. BD Biosciences GIBCO^® #16140), 3% SEAPLAQUE^® or other low-melting agarose in ddH₂O, sterile water, 50ml sterile conical screw-top tubes, and 37°C water bath microwave

[0263] Step one: Prepare infected monolayer of cells

1. Grow a suspension culture of Sf9 cells to a density of less than 3x10⁶.

2. Dilute this culture to a density of 5 to 6xlO⁵.

3. For a 6-well culture dish, transfer 2 ml of this cell suspension to each well. For 6 cm dishes, double all volumes in this protocol. Scale by surface area. This cell number will depend somewhat on the cell line and can be adjusted up or down according to your results. If there is no confluent monolayer by day 3, increase the cell number. There should be space available on Day 2.

4. Let the cells settle for at least 30 min to ensure the cells are firmly attached.

5. Meanwhile, dilute the plaque virus to 1 ml aliquots at dilutions of 10^~4, 10^~5, 10^~6 and 10^~7.

6. After SF cells have attached well to the plates, aspirate off the media.

7. Quickly add 1 ml of diluted virus to each well of the 6- well plate.

8. Transfer the plates to a rocking platform and slowly rock for at least two hours, four hours is preferred, though after this the benefit diminishes substantially. [0264] Step Two. Prepare the overlay agarose just before use.

9. Mix as follows: 1 part 2X Grace's Medium supplemented with 20% Fetal Calf Serum and 1 part

3% SEAPLAQUE^® Agarose in double distilled water (ddH₂O).

10. Melt the agarose completely.

11. Allow the agarose to cool slightly, to near 70⁰C and then aliquot 20 ml to each 50 ml conical tube.

12. Add 20 ml of room temp or warmer 2X Graces/FCS to each 20 ml aliquot of agarose, then place in a 35-37°C water bath.

13. Remove the overlay agarose from the water bath one tube at a time and check the temperature.

Let it cool to at least 38°C, but preferably less than 37°C. [0265] Step Three. Overlay agarose onto infected cell monolayer

14. When ready, aspirate all the medium off.

15. Return the plate to level and quickly add approximately 3 ml of molten overlay mix to each well by allowing it to slide down the far wall of the well and onto the plate.

16. After overlaying the cells let the plates sit level in the hood for 30 or so minutes to dry a bit and solidify.

17. Place in a 27°C, 98% humidity controlled incubator for at least 3 days. [0266] Step Four. Staining the Plates

18. Prepare a solution of 1% Neutral red.

19. Prepare an overlay agarose solution as above, but only prepare 1 ml for each assay.

20. Add 1/lOOth volume of the 1% Neutral Red solution to the molten agarose (e.g. 100 microliters to 10 ml).

21. Add approximately 1 ml of the Red Agarose to each well of a 6-well dish. Be sure the plate is level until the agarose sets up.

22. Add enough Red Agarose to cover the surface evenly.

23. Return the plate to the incubator for at least 4 hours. After several hours the plaques will begin to appear as clear spots among stained cells.

24. The plates can be left overnight before counting.

25. Controls can verify that longer incubations do not give higher titer results with the medium and cells used. [0267] In one embodiment, the positive plaques can be identified by end point dilution assay (EPDA). A 96-well plate EPDA can be used to replace the plaque assay and plaque purification as a method for either determining viral titer or identifying and purifying recombinant virus. A modified 12-well plate EPDA can be used as a routine method for determining viral titer; it is useful for estimating the efficiency of the initial co-transfection, identifying infected cells, approximating viral titers, and amplifying viral titer. In the 12-well EPDA, individual wells containing equal amounts of insect cells are inoculated with 100, 10, 1 or 0 μl aliquots of the original transfection supernatant, wild-type virus, or recombinant XyIE positive control viral supernatant. A visual comparison between cells in wells inoculated with 100, 10, 1 and 0 μl is used to estimate the viral titer.

[0268] For example, if cells receiving 100 μl of the initial co-transfection supernatant look infected in the EPDA, but cells receiving 10, 1 and 0 μl do not, then it is likely that the viral titer is low and should be amplified to produce a high titer stock. If wells receiving 100 μl of the original co-transfection supernatant look similar to those receiving 0 μl, it is likely that the original co-transfection did not result in a significant viral titer and must be repeated. When assaying the efficiency of a co-transfection or estimating the titer of a virus stock, if the EPDA shows a 10 fold decrease in the number of infected cells between dilutions, amplify the virus once or twice more to generate a high titer stock for protein production. However, if all three wells (100, 10, 1 μl) show equal signs of infection, the viral titer is high, ~ 2 x 10⁸ plaque -forming units (pfu) /ml. High titer recombinant virus stocks are used for infection of cells at optimal multiplicity of infection (MOI = # of virus / # of cells) resulting in maximum protein production.

[0269] If the EPDA is used as an amplification step to generate a high titer stock, cross contamination between wells containing different viruses, e. g., the highly infectious wild-type virus used as a positive control, can be avoided by using separate 12 well plates.

[0270] EPDA controls are recommended. The recombinant virus from a pVL1392-XylE transfection is a particularly useful positive control. Infected cells producing the the XyIE protein turn yellow in the presence of catechol and are easily identifiable. An example of a protocol for EPDA follows: [0271] Protocol

1. Dilute log-phase Sf 9 cells (with greater than 98% viability) to 1 x 105 cells /ml with fresh

TNM-FH medium. Seed 1 x 105 Sf9 cells per well on a 12-well plate (BD Falcon™, Cat. No. 353043). Allow cells to attach firmly, approximately 10 min. Confirm 30% confluency by visualization on a light microscope. Replace medium with 1 ml fresh TNM-FH.

2. Add 100, 10, 1 and 0 μl of the recombinant virus supernatant obtained 5 days after the start of co-transfection (or other virus stock), to separate wells. Do the same for the positive control, e.g., pVL1392-XylE supernatant.

3. Incubate the cells at 27°C for three days. Examine the cells for signs of infection.

4. A successful transfection should result in uniformly large infected cells in the 100, 10, and 1 μl experimental wells. 5. If only the 100 μl and 10 μl wells seem to have infected cells and the 1 μl well looks more like the control, the titer of virus supernatant is low. Amplify the virus an additional time before proceeding with protein production.

[0272] Protein production can be analyzed by western blot analysis (if antibodies are available) or by Coomassie blue-stained SDS-PAGE gel by harvesting cells from the 100 μl well and lysing in appropriate lysing buffer.

[0273] The virus supernatant from the 100 μl well can be kept as the first viral amplification stock, however care should be taken to avoid cross-contamination between wells containing different virus. [0274] To further purify the virus population, a plaque assay purification of the co-transfection supernatant can be performed using the approximate titer obtained from the EPDA. [0275] Once recombinant baculoviral vectors that express the proteins are established, then the virus can be amplified and purified for infection of SF cells.

[0276] Purification of Virus. Viral particles produced from the first passage are purified from the media using a known purification method such as sucrose density gradient centrifugation. For example, virus is harvested 24-48 hours post infection by centrifuging media of infected cells. The resulting viral pellet is resuspended in buffer and centrifuged through a buffered sucrose gradient. The virus band is harvested from the 40-45% sucrose region of the gradient, diluted with buffer and pelleted by centrifugation at 100,000xg. The purified virus pellet is resuspended in buffer and stored at -70⁰C or used in large scale infection of cells for protein production.

[0277] The infection process, including viral protein synthesis, viral assembly and partial cell lysis can be complete by approximately 72 hours post-infection. This can be protein dependent and thus can occur earlier or later. The proteins produced in infected cells can be radiolabeled with ³⁵S -methionine, ³H- leucine, or ³H-mannose and both cell-associated and cell-free polypeptides can be analyzed by electrophoresis on polyacrylamide gels to determine their molecular weight. The expression of these products can also be examined at different times post-infection, prior to cell lysis.

[0278] Immunological identification of expressed fusion polypeptides can be performed, e.g., by either direct immunoprecipitation or by Western blots. For Western blots, cell-associated proteins or the proteins in the media are separated on SDS polyacrylamide gels, transferred onto nitrocellulose or nylon filters, and identified with antiserum to the LF polypeptide or target antigen proteins or to the polyhedrin. Specifically bound antibody is detected by incubating the filters with ¹²⁵ 1-labeled protein A or enzyme conjugated anti-antibody, and followed by exposure to X-ray film at -80⁰C with intensifying screens or colorimetic reaction with enzyme substrate.

[0279] Having confirmed the identity of the expressed fusion polypeptides, the next step is to purify the proteins for uses and compositions described herein, e. g. evaluation for use as vaccines (e. g. protective/prophylactic or therapeutic vaccination) or screening agents. If the fusion polypeptides described herein are designed with secretion signal peptides, the encoded polypeptides are often released into the cell culture medium. Media from these infected cells can be concentrated and the proteins purified using standard methods. Salt precipitation, sucrose gradient centrifugation and chromatography, high or fast pressure liquid chromatography (HPLC or FPLC) can be used because these methods allow rapid, quantitative and large scale purification of proteins, and do not denature expressed products. [0280] The efficiency of synthesis of the desired gene product is dependent on multiple factors including: (1) the choice of an expression vector system; (2) the number of gene copies that will be available in the cells as templates for the production of mRNA; (3) the promoter strength; (4) the stability and structure of the mRNA; (5) the efficient binding of ribosomes for the initiation or translation; (6) the properties of the protein product, such as, the stability of the gene product or lethality of the product to the host cells; and (7) the ability of the system to synthesize and export the protein from the cells, thus simplifying subsequent analysis, purification and use.

[0281] Purification of recombinant influenza proteins expressed in a BEVS is known in the art, for example, in U. S. Patent Nos. 5,290,686, 5,976,552, 7,399,840 and U. S. Patent Application No. 2008/0008725, all of which are incorporated herein by reference in their entirety.

Production of fusion polypeptide using other expression systems

[0282] The fusion polypeptides described herein can all be synthesized and purified by protein and molecular methods that are well known to one skilled in the art. Preferably molecular biology methods and recombinant heterologous protein expression systems are used. For example, recombinant protein can be expressed in mammalian, insect, yeast, or plant cells.

[0283] Some examples of recombinant cloning and truncation of LF, LFn, their expression, and specific site mutations and insertions are described, for example, in WO/2002/079417, WO/2008/048289, U. S. Patent Application No. 2004/0166120, Huyen Cao, et. al., 2002, J. Infectious Diseases; 185:244-251; N. Kushner, et. al., 2003, Proc. Natl. Acad. Sci. U S A. 100:6652-6657; Ballard, J. D., et. al., 1996, Proc. Natl Acad. Sci. USA 93:12531-12534; and Goletz, T. J. et al., 1997, Proc. Natl. Acad. Sci. USA 94: 12059-12064, all of which are incorporated herein by reference in their entirety. Approaches similar to those described in these references can be used to produce the fusion polypeptides as described herein. [0284] In one embodiment, provided herein is an isolated polynucleotide encoding a fusion polypeptide or a non-fusion polypeptide described herein. Conventional polymerase chain reaction (PCR) cloning techniques can be used to construct a chimeric or fusion coding sequence encoding a fusion polypeptide as described herein. A coding sequence can be cloned into a general purpose cloning vector such as pUC19, pBR322 , pBLUESCRIPT^® vectors (STRATAGENE^® Inc.) or pCR TOPO^® from INVITROGEN™ Inc. The resultant recombinant vector carrying the nucleic acid encoding a polypeptide as described herein can then be used for further molecular biological manipulations such as site-directed mutagenesis to create a variant fusion polypeptide as described herein or can be subcloned into protein expression vectors or viral vectors for protein synthesis in a variety of protein expression systems using host cells selected from the group consisting of mammalian cell lines, insect cell lines, yeast, bacteria, and plant cells.

[0285] Each PCR primer should have at least 15 nucleotides overlapping with its corresponding templates at the region to be amplified. The polymerase used in the PCR amplification should have high fidelity such as STRAT AGENE^® P/wULTRA^® polymerase for reducing sequence mistakes during the PCR amplification process. For ease of ligating several separate PCR fragments together, for example in the construction of a fusion polypeptide, and subsequently inserting into a cloning vector, the PCR primers should also have distinct and unique restriction digestion sites on their flanking ends that do not anneal to the DNA template during PCR amplification. The choice of the restriction digestion sites for each pair of specific primers should be such that the fusion polypeptide coding DNA sequence is in- frame and will encode the fusion polypeptide from beginning to end with no stop codons. At the same time the chosen restriction digestion sites should not be found within the coding DNA sequence for the fusion polypeptide. The coding DNA sequence for the intended polypeptide can be ligated into cloning vector pBR322 or one of its derivatives, for amplification, verification of fidelity and authenticity of the chimeric coding sequence, substitutions/or specific site-directed mutagenesis for specific amino acid mutations and substitutions in the polypeptide.

[0286] Alternatively the coding DNA sequence for the polypeptide can be PCR cloned into a vector using for example, INVITROGEN™ Inc.'s TOPO^® cloning method comprising topoisomerase-assisted TA vectors such as pCR^®-TOPO, pCR^®-Blunt II-TOPO, pENTR/D-TOPO®, and pENTR/SD/D-TOPO^®. Both pENTR/D-TOPO^®, and pENTR/SD/D-TOPO^® are directional TOPO entry vectors which allow the cloning of the DNA sequence in the 5'→3' orientation into a GATEWAY® expression vector. Directional cloning in the 5'→3' orientation facilitates the unidirectional insertion of the DNA sequence into a protein expression vector such that the promoter is upstream of the 5' ATG start codon of the fusion polypeptide coding DNA sequence, enabling promoter driven protein expression. The recombinant vector carrying the coding DNA sequence for the fusion polypeptide can be transfected into and propagated in general cloning E. coli such as XLIBlue, SURE^® (STRATAGENE^®) and TOP-IO cells (INVITROGEN™ Inc.).

[0287] Standard techniques known to those of skill in the art can be used to introduce mutations (to create amino acid substitutions in the polypeptide sequence of the fusion polypeptide described herein, e. g., in the LFn polypeptide, i. e. SEQ. ID. No. 3 or 4 or 5) in the nucleotide sequence encoding the fusion polypeptide described herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, the variant fusion polypeptide has less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the fusion polypeptides described herein.

[0288] Certain silent or neutral missense mutations can also be made in the DNA coding sequence that do not change the encoded amino acid sequence or the capability to promote transmembrane delivery. These types of mutations are useful to optimize codon usage, or to improve recombinant protein expression and production. [0289] Specific site -directed mutagenesis of a coding sequence for the fusion polypeptide in a vector can be used to create specific amino acid mutations and substitutions. Site -directed mutagenesis can be carried out using, e. g. the QUIKCHANGE^® site -directed mutagenesis kit from Stratagene according to the manufacturer's instructions.

[0290] In one embodiment, described herein are expression vectors comprising the coding DNA sequence for the polypeptides described herein for the expression and purification of the recombinant polypeptide produced from a protein expression system using host cells selected from, e.g., mammalian, insect, yeast, or plant cells. The expression vector should have the necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences, ribosome recognition and TATA (SEQ. ID. No. 33) box, and 3' UTR AAUAAA (SEQ. ID. No. 34) transcription termination sequence for efficient gene transcription and translation in its respective host cell. The expression vector is, preferably, a vector having the transcription promoter selected from a group consisting of CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, β-actin promoter, SV40 (simian virus 40) promoter and muscle creatine kinase promoter, and the transcription terminator selected from a group consisting of SV40 poly (A) and BGH terminator; more preferably, an expression vector having the early promoter/enhancer sequence of cytomegalovirus and the adenovirus tripartite leader/intron sequence and containing the replication orgin and poly (A) sequence of SV40. The expression vector can have additional sequence such as 6X-histidine, V5, thioredoxin, glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose binding peptide, metal-binding peptide, HA and "secretion" signals (Honeybee melittin, α-factor, PHO, Bip), which are incorporated into the expressed fusion polypeptide. In addition, there can be enzyme digestion sites incorporated after these sequences to facilitate enzymatic removal of them after they are not needed. These additional sequences are useful for the detection of fusion polypeptide expression, for protein purification by affinity chromatography, enhanced solubility of the recombinant protein in the host cytoplasm, and/or for secreting the expressed fusion polypeptide out into the culture media or the spheroplast of the yeast cells. The expression of the fusion polypeptide can be constitutive in the host cells or it can be induced, e.g., with copper sulfate, sugars such as galactose, methanol, methylamine, thiamine, tetracycline, infection with baculo virus, and (isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analog of lactose.

[0291] In another embodiment, the expression vector comprising a polynucleotide described herein is a viral vector, such as adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus vectors, among others. Recombinant viruses provide a versatile system for gene expression studies and therapeutic applications.

[0292] The polypeptides described herein can be expressed in a variety of expression host cells e.g., yeasts, mammalian cells, insect cells and plant cells such as Chlamadomonas, or even in cell-free expression systems. From the cloning vector, the nucleic acid can be subcloned into a recombinant expression vector that is appropriate for the expression of fusion polypeptide in mammalian, insect, yeast, or plant cells or a cell-free expression system such as a rabbit reticulocyte expression system. Some vectors are designed to transfer coding nucleic acid for expression in mammalian cells, insect cells and year in one single recombination reaction. For example, some of the GATEWAY^® (INVITROGEN™ Inc.) destination vectors are designed for the construction of baculo virus, adenovirus, adeno-associated virus (AAV), retrovirus, and lentiviruses, which upon infecting their respective host cells, permit heterologous expression of fusion polypeptides in the appropriate host cells. Transferring a gene into a destination vector is accomplished in just two steps according to manufacturer's instructions. There are GATEWAY^® expression vectors for protein expression in insect cells, mammalian cells, and yeast. Following transformation and selection in E. coli, the expression vector is ready to be used for expression in the appropriate host.

[0293] Examples of other expression vectors and host cells are the strong CMV promoter-based pcDNA3.1 (INVITROGEN™ Inc.) and pCINEO vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral vector vectors pADENO-X™, pAd5F35, pLP-ADENO™-X-CMV (CLONTECH^®), pAd/CMV/V5-DEST, pAd-DEST vector (INVITROGEN™ Inc. ) for adenovirus-mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with the RETRO-X™ system from Clontech for retro viral-mediated gene transfer and expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (INVITROGEN™ ) for lenti virus-mediated gene transfer and expression in mammalian cells; adeno virus-associated virus expression vectors such as pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associated virus-mediated gene transfer and expression in mammalian cells; BACpakβ baculo virus (Clontech) and pFASTBAC™ HT (INVITROGEN™ ) for the expression in S.frugiperda 9 (Sf9), SfI l, Tn-368 and BTI-TN-5B4-1 insect cell lines; pMT/BiP/V5-His (INVITROGEN™ ) for the expression in Drosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ, pFLDα and pFLD (INVITROGEN™ ) for expression in Pichia pastoris and vectors pMETα and pMET for expression in P. methanolica; pYES2/GS and pYDl (INVITROGEN™ ) vectors for expression in yeast S. cerevisiae. Recent advances in the large scale expression heterologous proteins in Chlamydomonas reinhardtii are described by Griesbeck C. et. al., 2006 MoI. Biotechnol. 34:213-33 and Fuhrmann M., 2004, Methods MoI Med. 94: 191-5. Foreign heterologous coding sequences are inserted into the genome of the nucleus, chloroplast and mitochondria by homologous recombination. The chloroplast expression vector p64 carrying the most versatile chloroplast selectable marker aminoglycoside adenyl transferase (aadA), which confer resistance to spectinomycin or streptomycin, can be used to express foreign protein in the chloroplast. The biolistic gene gun method can be used to introduce the vector in the algae. Upon its entry into chloroplasts, the foreign DNA is released from the gene gun particles and integrates into the chloroplast genome through homologous recombination.

[0294] In some embodiments, the fusion polypeptides described herein are expressed from viral infection of mammalian cells. The viral vectors can be, for example, adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. A simplified system for generating recombinant adenoviruses is presented by He et al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. The gene of interest is first cloned into a shuttle vector, e.g. pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pADEASY-1 of STRAT AGENE^®'s ADEASY™ Adenoviral Vector System. Recombinant adenovirus vectors are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, for example HEK 293 cells (El -transformed human embryonic kidney cells) or 911 (El -transformed human embryonic retinal cells) (Human Gene Therapy 7:215-222, 1996). Recombinant adenovirus are generated within the HEK 293 cells. [0295] Recombinant lentivirus has the advantage of delivery and expression of fusion polypeptides in dividing and non-dividing mammalian cells. The HIV-I based lentivirus can effectively transduce a broader host range than the Moloney Leukemia Virus (MoMLV) -based retroviral systems. Preparation of the recombinant lentivirus can be achieved using, for example, the pLenti4/V5-DEST™, pLenti6/V5- DEST™ or pLenti vectors together with VIRAPOWER™ Lentiviral Expression systems from INVITROGEN™ Inc.

[0296] Recombinant adeno-associated virus (rAAV) vectors are applicable to a wide range of host cells including many different human and non-human cell lines or tissues. rAAVs are capable of transducing a broad range of cell types and transduction is not dependent on active host cell division. High titers, > 10⁸ viral particle/ml, are easily obtained in the supernatant and 10¹¹ -10¹² viral particle/ml with further concentration. The transgene is integrated into the host genome so expression is long term and stable. [0297] Large scale preparation of AAV vectors is made by a three-plasmid cotransfection of a packaging cell line: AAV vector carrying the coding nucleic acid, AAV RC vector containing AAV rep and cap genes, and adenovirus helper plasmid pDF6, into 50 x 150 mm plates of subconfluent 293 cells. Cells are harvested three days after transfection, and viruses are released by three freeze-thaw cycles or by sonication.

[0298] AAV vectors can be purified by two different methods depending on the serotype of the vector. AAV2 vector is purified by the single-step gravity-flow column purification method based on its affinity for heparin (Auricchio, A., et. al., 2001, Human Gene therapy 12:71-6; Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford, C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5 vectors are currently purified by three sequential CsCl gradients.

[0299] The polypeptides described herein can be expressed and purified by a variety methods known to one skilled in the art, for example, the fusion polypeptides described herein can be purified from any suitable expression system. Fusion polypeptides can be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al. supra). [0300] A number of procedures can be employed when recombinant proteins are purified. For example, proteins having established molecular adhesion properties can be reversibly fused to the protein of choice. With the appropriate ligand, the protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, the protein of choice can be purified using affinity or immunoaffinity columns. [0301] After the protein is expressed in the host cells, the host cells can be lysed to liberate the expressed protein for purification. Methods of lysing the various host cells are featured in "Sample Preparation-Tools for Protein Research" EMD Bioscience and in the Current Protocols in Protein Sciences (CPPS). A preferred purification method is affinity chromatography such as metal-ion affinity chromatograph using nickel, cobalt, or zinc affinity resins for histidine-tagged fusion polypeptides. Methods of purifying histidine-tagged recombinant proteins are described by Clontech using their TALON^® cobalt resin and by NOVAGEN^® in their pET system manual, 10th edition. Another preferred purification strategy is immuno-affinity chromatography, for example, anti-myc antibody conjugated resin can be used to affinity purify myc-tagged fusion polypeptides. When appropriate protease recognition sequences are present, fusion polypeptides can be cleaved from the histidine or myc tag, releasing the fusion polypeptide from the affinity resin while the histidine-tags and myc-tags are left attached to the affinity resin.

[0302] Standard protein separation techniques for purifying recombinant and naturally occurring proteins are well known in the art, e. g. solubility fractionation, size exclusion gel filtration, and various column chromatography.

[0303] Solubility fractionation: Often as an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

[0304] Size exclusion filtration: The molecular weight of the protein of choice can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, AMICON^® or MILLIPORE^® membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.

[0305] Column chromatography: The protein of choice can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands. In addition, antibodies raised against recombinant or naturally occurring proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech). For example, LFn can be purified using a PA63 heptamer affinity column (Singh et al., 1994, J. Biol. Chem. 269:29039-29046). [0306] In some embodiments, a combination of purification steps comprising, for example: (i) anion exchange chromatography, (ii) hydroxyapatite chromatography, (iii) hydrophobic interaction chromatography, and (iv) size exclusion chromatography can be used to purify the fusion polypeptides described herein.

[0307] Cell-free expression systems are also contemplated. Cell-free expression systems offer several advantages over traditional cell-based expression methods, including the easy modification of reaction conditions to favor protein folding, decreased sensitivity to product toxicity and suitability for high- throughput strategies such as rapid expression screening or large amount protein production because of reduced reaction volumes and process time. The cell-free expression system can use plasmid or linear DNA. Moreover, improvements in translation efficiency have resulted in yields that exceed a milligram of protein per milliliter of reaction mix. Commercially available cell-free expression systems include the TNT coupled reticulocyte lysate Systems (Promega) which uses rabbit reticulocyte-based in-vitro system.

Formulation and Application

[0308] In one embodiment, provided herein is a method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising, or alternatively, consisting essentially of an inactivated influenza virus and an influenza virus NP polypeptide. Alternatively, the influenza virus in an attenuated influenza virus. Alternatively, the inactivated or attenuated influenza virus is replaced with isolated full-length influenza virus HA and/or NA polypeptide.

[0309] In another embodiment, provided herein is a method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising, or alternatively, of an inactivated influenza virus, an influenza virus NP polypeptide, and an influenza virus matrix (M) protein. Alternatively, the influenza virus in an attenuated influenza virus. Alternatively, the inactivated or attenuated influenza virus is replaced with isolated full-length influenza virus HA and/or NA polypeptide.

[0310] In another embodiment, provided herein is a method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising a pharmaceutically acceptable carrier, a B. anthracis Lethal Factor (LF) polypeptide and an antigen preparation, the antigen preparation comprising an inactivated or attenuated influenza virus or isolated full-length influenza virus HA and/or NA polypeptide, and an influenza virus nucleoprotein (NP) polypeptide. In yet another embodiment, the antigen preparation further comprises an influenza virus matrix (M) polypeptide.

[0311] In one embodiment, the vaccine compositions described herein comprise a polypeptide that is expressed and purified from insect cells. In one embodiment, the vaccine composition comprises a plurality of polypeptides that are expressed and purified from insect cells. In another embodiment, the vaccine composition comprises an LF polypeptide, wherein the LF polypeptide is N-glycosylated. The N-glycosylation can be at asparagine 62, 212 and/or 286.

[0312] In one embodiment, the vaccine compositions described herein comprise a pharmaceutically acceptable carrier. In another embodiment, the vaccine composition described herein is formulated for administering to a mammal. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference.

[0313] In one embodiment, the vaccine compositions described herein comprise pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol. For all administrations, conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained release preparations. For examples of sustained release compositions, see U.S. Patent Nos. 3,773,919, 3,887,699, EP 58,481A, EP 158,277A, Canadian Patent No. 1176565; U. Sidman et al., Biopolymers 22:547 (1983) and R. Langer et al., Chem. Tech. 12:98 (1982). The proteins will usually be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml per application per patient. [0314] In one embodiment, other ingredients can be added to vaccine formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol. [0315] In one embodiment, the vaccine compositions described herein for administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).

[0316] In some embodiments, the vaccine composition described herein further comprises pharmaceutical excipients including, but not limited to biocompatible oils, physiological saline solutions, preservatives, carbohydrate, protein , amino acids, osmotic pressure controlling agents, carrier gases, pH- controlling agents, organic solvents, hydrophobic agents, enzyme inhibitors, water absorbing polymers, surfactants, absorption promoters and anti-oxidative agents. Representative examples of carbohydrates include soluble sugars such as hydropropyl cellulose, carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid, chitosan, alginate, glucose, xylose, galactose, fructose, maltose, saccharose, dextran, chondroitin sulfate, etc. Representative examples of proteins include albumin, gelatin, etc. Representative examples of amino acids include glycine, alanine, glutamic acid, arginine, lysine, and their salts.

[0317] In some embodiments, the polypeptides described herein can be solubilized in water, a solvent such as methanol, or a buffer. Suitable buffers include, but are not limited to, phosphate buffered saline Ca ²VMg²⁺ free (PBS), normal saline (150 mM NaCl in water), and Tris buffer. Antigen not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS. In the case of antigen soluble only at acid pH, acetate-PBS at acid pH may be used as a diluent after solubilization in dilute acetic acid. Glycerol can be a suitable non-aqueous buffer for use in the present invention.

[0318] If the polypeptide is not soluble per se, the polypeptide can be present in the formulation in a suspension or even as an aggregate. In some embodiments, hydrophobic antigen can be solubilized in a detergent, for example a polypeptide containing a membrane-spanning domain. Furthermore, for formulations containing liposomes, an antigen in a detergent solution (e.g., a cell membrane extract) may be mixed with lipids, and liposomes then may be formed by removal of the detergent by dilution, dialysis, or column chromatography.

[0319] In some embodiments, the vaccine composition is administered in combination with other therapeutic ingredients including, e.g., γ-interferon, cytokines, chemotherapeutic agents, or antiinflammatory or anti- viral agents.

[0320] In some embodiments, the vaccine composition is administered in a pure or substantially pure form, but it is preferable to present it as a pharmaceutical composition, formulation or preparation. Such formulation comprises polypeptides described herein together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. Other therapeutic ingredients include compounds that enhance antigen presentation, e.g., gamma interferon, cytokines, chemotherapeutic agents, or anti-inflammatory agents. The formulations can conveniently be presented in unit dosage form and may be prepared by methods well known in the pharmaceutical art. For example, Plotkin and Mortimer (In 'Vaccines', 1994, W.B. Saunders Company; 2nd edition) describes vaccination of animals or humans to induce an immune response specific for particular pathogens, as well as methods of preparing antigen, determining a suitable dose of antigen, and assaying for induction of an immune response.

[0321] In some embodiments, the vaccine composition described herein further comprises an adjuvant. Adjuvants are a heterogeneous group of substances that enhance the immunological response against an antigen that is administered simultaneously. In some instances, adjuvants are added to a vaccine to improve the immune response so that less vaccine is needed. Adjuvants serve to bring the antigen — the substance that stimulates the specific protective immune response — into contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (magnitude or duration). Adjuvants can also decrease the toxicity of certain antigens; and provide solubility to some vaccine components. Almost all adjuvants used today for enhancement of the immune response against antigens are particles or form particles together with the antigen. In the book "Vaccine Design — the subunit and adjuvant approach" (Ed: Powell & Newman, Plenum Press, 1995) almost all known adjuvants are described both regarding their immunological activity and regarding their chemical characteristics. The type of adjuvants that do not form particles are a group of substances that act as immunological signal substances and that under normal conditions consist of the substances that are formed by the immune system as a consequence of the immunological activation after administration of particulate adjuvant systems.

[0322] Using particulate systems as adjuvants, the antigens are associated or mixed with or into a matrix, which has the characteristics of being slowly biodegradable. Care must be taken to ensure that that the matrices do not form toxic metabolites. Preferably, the main kinds of matrices used are mainly substances originating from a body. These include lactic acid polymers, poly-amino acids (proteins), carbohydrates, lipids and biocompatible polymers with low toxicity. Combinations of these groups of substances originating from a body or combinations of substances originating from a body and biocompatible polymers can also be used. Lipids are the preferred substances since they display structures that make them biodegradable as well as the fact that they are a critical element in all biological membranes.

[0323] Adjuvants for vaccines are well known in the art. Examples include, but not limited to, monoglycerides and fatty acids (e. g. a mixture of mono-olein, oleic acid, and soybean oil); mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion); particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG); microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC- 529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self organize into liposomes), OM- 174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects); endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and inert vehicles, such as gold particles. Newer adjuvants are described in U. S. Patent No. 6,890,540, U. S.

Patent Application No. 20050244420, and PCT/SE97/01003, the contents of which are incorporated herein by reference in their entirety.

[0324] Formulations suitable for intravenous, intramuscular, intranasal, oral, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g., 0.1-2.0 M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering the solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampoules or vials.

[0325] Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U. S. Patent

No. 4,522,811.

[0326] Formulations for an intranasal delivery are described in US Patent Nos. 5,427,782, 5,843,451 and 6,398,774.

[0327] The formulations of the vaccine compositions can incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharide, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. Two or more stabilizers may be used in aqueous solutions at the appropriate concentration and/or pH. The specific osmotic pressure in such aqueous solution is generally in the range of 0.1-3.0 osmoses, preferably in the range of 0.80-1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8.

[0328] When oral preparations are desired, the vaccine compositions can be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.

[0329] A method of immunization or vaccinating a mammal against an influenza virus comprises administering a vaccine composition described herein.

[0330] In some embodiments, the vaccine compositions described herein can be administered intravenously, intranasally, intramuscularly, subcutaneously, infraperitoneally or orally. A preferred route of administration is oral, intranasal or intramuscular.

[0331] Vaccination can be conducted by conventional methods. For example, a polypeptide can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. The vaccine can be administered by any route appropriate for eliciting an immune response. The vaccine can be administered once or at periodic intervals until an immune response is elicited. Immune responses can be detected by a variety of methods known to those skilled in the art, including but not limited to, antibody production, cytotoxicity assay, proliferation assay and cytokine release assays. For example, samples of blood can be drawn from the immunized mammal, and analyzed for the presence of antibodies against the NP, Ml, and/or M2 proteins by ELISA (see de Boer GF, et. al., 1990, Arch Virol. 115:47-61) (e. g. using The

ImmTech Influenza A Nucleoprotein Antigen Capture ELISA kits (IAV-1192 and IVA- 1480) and the titer of these antibodies can be determined by methods known in the art.

[0332] The precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. For example, a range of 25 μg - 900 μg total protein can be administered intradermally, monthly for 3 months.

[0333] Ultimately, the attending physician will decide the amount of protein or vaccine composition to administer to particular individuals.

[0334] The present invention can be defined in any of the following alphabetized paragraphs:

[A] A vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide.

[B] A vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide and an influenza virus matrix (M) protein.

[C] The vaccine composition of paragraph [A] or [B], wherein the influenza virus polypeptide is expressed and isolated from a bacculovirus expression system.

[D] The vaccine composition of paragraph [C], wherein the influenza virus polypeptide is fused a Bacillus anthracis Lethal Factor (LF) polypeptide.

[E] The vaccine of paragraph [D], wherein the LF polypeptide is a LFn polypeptide.

[F] The vaccine composition of paragraph [A] or [B], wherein said composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD50 of H5N2 influenza virus.

[G] The vaccine composition of paragraph [A] or [B], wherein said antigen consists of the influenza virus NP polypeptide of SEQ. ID. No. 6, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[H] The vaccine composition of paragraph [B], wherein said antigen consists of the influenza virus Ml polypeptide of SEQ. ID. No. 7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[I] The vaccine composition of paragraph [B], wherein said antigen consists of the influenza virus M2 polypeptide of SEQ. ID. No. 8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[J] The vaccine composition of paragraph [A] or [B] further comprising an adjuvant. [K] The vaccine composition of paragraph [J] , wherein said adjuvant is selected from the group consisting of QS- 21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005,

GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026,

Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

[L] The vaccine composition of paragraph [A] or [B], wherein said influenza virus is selected from strains consisting of influenza virus A H3N2, HlNl, H7N2, H7N3, Hl, H5N1,

H1N2, H9N2, influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia,

B/Shandong, and B/Hong Kong.

[M] A method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising an influenza virus NP polypeptide.

[N] A method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising an influenza virus NP polypeptide and an influenza virus matrix (M) protein.

[O] The method of paragraph [M] or [N], wherein said influenza virus is selected from strains consisting of influenza virus A H3N2, HlNl, H7N2, H7N3, Hl, H5N1, H1N2, H9N2, influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, and

B/Hong Kong.

[P] The method of paragraph [M] or [N], wherein said influenza virus NP polypeptide is a polypeptide of SEQ. ID. No. 6, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[Q] The method of paragraph [N], wherein said influenza virus M polypeptide is a Ml polypeptide of SEQ. ID. No. 7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[R] The method of paragraph [N], wherein said influenza virus M polypeptide is a M2 polypeptide of SEQ. ID. No. 8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[S] The method of paragraph [M] or [N], wherein said vaccine composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD50 of H5N2 influenza virus.

[T] The method of paragraph [M] or [N], wherein the influenza virus polypeptide is expressed and isolated from a bacculovirus expression system.

[U] The method of paragraph [T], wherein the influenza virus polypeptide is fused a Bacillus anthracis Lethal Factor (LF) polypeptide.

[V] The method of paragraph [U], wherein the LF polypeptide is a LFn polypeptide.

[W] A vaccine composition comprising a pharmaceutically acceptable carrier, a Bacillus anthracis Lethal Factor (LF) polypeptide and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide. [X] The vaccine composition of paragraph [W], the antigen preparation further comprising an influenza virus matrix (M) polypeptide.

[Y] The vaccine composition of paragraph [W] or [X] , wherein said LF polypeptide is an N- terminal (LFn) polypeptide, or conservative substitution variant thereof, that promotes transmembrane delivery to the cytosol of an intact cell.

[Z] The vaccine composition of paragraph [Y], wherein said LFn polypeptide is N- glycosylated.

[AA] The vaccine composition of paragraph [Y], wherein said LFn polypeptide comprises at least the 60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

[BB] The vaccine composition of paragraph [Y], wherein said LFn polypeptide comprises at least the 80 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

[CC] The vaccine composition of paragraph [Y], wherein said LFn polypeptide comprises at least the 104 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

[DD] The vaccine composition of paragraph [Y], wherein said LFn polypeptide comprises the amino acid sequence corresponding to SEQ. ID. No. 5, or a conservative substitution variant thereof.

[EE] The vaccine composition of paragraph [Y], wherein said LFn polypeptide does not bind

B. anthracis protective antigen protein.

[FF] The vaccine composition of paragraph [Y], wherein said LFn polypeptide substantially lacks the amino acids 1-33 of SEQ. ID. No. 3.

[GG] The vaccine composition of paragraph [Y], wherein said LFn polypeptide consists of

SEQ. ID. No. 5, or a conservative substitution variant thereof.

[HH] The vaccine composition of paragraph [X], wherein said influenza virus M polypeptide is a Ml polypeptide of SEQ. ID. No. 7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[II] The vaccine composition of paragraph [X], wherein said influenza virus M polypeptide is a M2 polypeptide of SEQ. ID. No. 8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

[JJ] The vaccine composition of any of paragraphs [W]-[II] , wherein said LF polypeptide is fused to said influenza virus NP polypeptide or a fragment thereof of at least 15 amino acids.

[KK] The vaccine composition of any of paragraphs [X]-[II], wherein said LF polypeptide is fused to said influenza virus M polypeptide or a fragment thereof of at least 15 amino acids.

[LL] The vaccine composition of any of paragraphs [W]-[II] , wherein said influenza virus is selected from strains consisting of influenza virus A H3N2, HlNl, H7N2, H7N3, Hl, H5N1, H1N2, H9N2, influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, and B/Hong Kong.

[MM] A method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition of any of paragraphs [W]-[LL], to a said mammal. [NN] The method of paragraph [MM] wherein said vaccine composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD50 of influenza virus having same HA/NA serotypes as the source of influenza virus- derived components in the vaccine composition.

[OO] The method of paragraph [MM] wherein said vaccine composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD50 of H5N2 influenza virus.

[0335] The contents of all references cited throughout this application are incorporated herein by reference.

Claims

What is claimed:

1. A vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide.

2. A vaccine composition comprising a pharmaceutically acceptable carrier and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide and an influenza virus matrix (M) protein.

3. The vaccine composition of claim 1 or 2, wherein the influenza virus polypeptide is expressed and isolated from a bacculovirus expression system.

4. The vaccine composition of claim 3, wherein the influenza virus polypeptide is fused a

Bacillus anthracis Lethal Factor (LF) polypeptide.

5. The vaccine of claim 4, wherein the LF polypeptide is a LFn polypeptide.

6. The vaccine composition of claim 1 or 2, wherein said composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD₅₀ of H5N2 influenza virus.

7. The vaccine composition of claim 1 or 2, wherein said antigen consists of the influenza virus

NP polypeptide of SEQ. ID. No. 6, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

8. The vaccine composition of claim 2, wherein said antigen consists of the influenza virus Ml polypeptide of SEQ. ID. No. 7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

9. The vaccine composition of claim 2, wherein said antigen consists of the influenza virus M2 polypeptide of SEQ. ID. No. 8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

10. The vaccine composition of claim 1 or 2 further comprising an adjuvant.

11. The vaccine composition of claim 10, wherein said adjuvant is selected from the group consisting of QS- 21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

12. The vaccine composition of claim 1 or 2, wherein said influenza virus is selected from strains consisting of influenza virus A H3N2, HlNl, H7N2, H7N3, Hl, H5N1, H1N2, H9N2, influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, and B/Hong Kong.

13. A method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising an influenza virus NP polypeptide.

14. A method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition comprising, as a target antigen, a preparation comprising an influenza virus NP polypeptide and an influenza virus matrix (M) protein.

15. The method of claim 13 or 14, wherein said influenza virus is selected from strains consisting of influenza virus A H3N2, HlNl, H7N2, H7N3, Hl, H5N1, H1N2, H9N2, influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, and B/Hong Kong.

16. The method of claim 13 or 14, wherein said influenza virus NP polypeptide is a polypeptide of SEQ. ID. No. 6, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

17. The method of claim 14, wherein said influenza virus M polypeptide is a Ml polypeptide of

SEQ. ID. No. 7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

18. The method of claim 14, wherein said influenza virus M polypeptide is a M2 polypeptide of

SEQ. ID. No. 8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

19. The method of claim 13 or 14, wherein said vaccine composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD50 of H5N2 influenza virus.

20. The method of claim 13 or 14, wherein the influenza virus polypeptide is expressed and isolated from a bacculovirus expression system.

21. The method of claim 20, wherein the influenza virus polypeptide is fused a Bacillus anthracis Lethal Factor (LF) polypeptide.

22. The method of claim 21 , wherein the LF polypeptide is a LFn polypeptide.

23. A vaccine composition comprising a pharmaceutically acceptable carrier, a Bacillus anthracis Lethal Factor (LF) polypeptide and an antigen preparation, the antigen preparation comprising an influenza virus nucleoprotein (NP) polypeptide.

24. The vaccine composition of claim 23, the antigen preparation further comprising an influenza virus matrix (M) polypeptide.

25. The vaccine composition of claim 23 or 24, wherein said LF polypeptide is an N-terminal

(LFn) polypeptide, or conservative substitution variant thereof, that promotes transmembrane delivery to the cytosol of an intact cell.

26. The vaccine composition of claim 25, wherein said LFn polypeptide is N-glycosylated.

27. The vaccine composition of claim 25, wherein said LFn polypeptide comprises at least the

60 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

28. The vaccine composition of claim 25, wherein said LFn polypeptide comprises at least the

80 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

29. The vaccine composition of claim 25, wherein said LFn polypeptide comprises at least the

104 carboxy-terminal amino acids of SEQ. ID. No. 3, or a conservative substitution variant thereof.

30. The vaccine composition of claim 25, wherein said LFn polypeptide comprises the amino acid sequence corresponding to SEQ. ID. No. 5, or a conservative substitution variant thereof.

31. The vaccine composition of claim 25, wherein said LFn polypeptide does not bind B. anthracis protective antigen protein.

32. The vaccine composition of claim 25, wherein said LFn polypeptide substantially lacks the amino acids 1-33 of SEQ. ID. No. 3.

33. The vaccine composition of claim 25, wherein said LFn polypeptide consists of SEQ. ID.

No. 5, or a conservative substitution variant thereof.

34. The vaccine composition of claim 24, wherein said influenza virus M polypeptide is a Ml polypeptide of SEQ. ID. No. 7, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

35. The vaccine composition of claim 24, wherein said influenza virus M polypeptide is a M2 polypeptide of SEQ. ID. No. 8, a conservative substitution mutant thereof or a fragment of at least 15 amino acids thereof.

36. The vaccine composition of any of claims 23-35, wherein said LF polypeptide is fused to said influenza virus NP polypeptide or a fragment thereof of at least 15 amino acids.

37. The vaccine composition of any of claims 24-35, wherein said LF polypeptide is fused to said influenza virus M polypeptide or a fragment thereof of at least 15 amino acids.

38. The vaccine composition of any of claims 23-35, wherein said influenza virus is selected from strains consisting of influenza virus A H3N2, HlNl, H7N2, H7N3, Hl, H5N1, H1N2, H9N2, influenza B virus B/Yamagata, B/Sichuan, B/Victoria, B/Brisbane, B/Malaysia, B/Shandong, and B/Hong Kong.

39. A method of vaccinating a mammal against an influenza virus, the method comprising administering a vaccine composition of any of claims 23-38 to a said mammal.

40. The method of claim 39 wherein said vaccine composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD₅₀ of influenza virus having same HA/NA serotypes as the source of influenza virus-derived components in the vaccine composition.

41. The method of claim 39 wherein said vaccine composition, when administered to mice, provokes an immune response that prevents death in at least 20% of animals challenged with 5 LD₅₀ of H5N2 influenza virus.