Influenza virus vaccine composition and methods of use (26-May-2009)

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. application Ser. No. 11/131,479, filed May 18, 2005, which claims the benefit of U.S. Provisional Application No. 60/571,854, filed May 18, 2004, both of which are incorporated herein by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

This application includes a “SequenceListing.txt”, 334,953 bytes, created on Jun. 25, 2008 and submitted electronically via EFS-Web which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to influenza virus vaccine compositions and methods of treating or preventing influenza infection and disease in mammals. Influenza is an acute febrile illness caused by infection of the respiratory tract. There are three types of influenza viruses: A, B, and C “IAV,” “IBV” or “IAC,” respectively, or generally “IV”. Type A, which includes several subtypes, causes widespread epidemics and global pandemics such as those that occurred in 1918, 1957 and 1968. Type B causes regional epidemics. Type C causes sporadic cases and minor, local outbreaks. These virus types are distinguished in part on the basis of differences in two structural proteins, the nucleoprotein, found in the center of the virus, and the matrix protein, which forms the viral shell.

The disease can cause significant systemic symptoms, severe illness requiring hospitalization (such as viral pneumonia), and complications such as secondary bacterial pneumonia. More than 20 million people died during the pandemic flu season of 1918/1919, the largest pandemic of the 20^th century. Recent epidemics in the United States are believed to have resulted in greater than 10,000 (up to 40,000) excess deaths per year and 5,000-10,000 deaths per year in non-epidemic years.

The best strategy for prevention of morbidity and mortality associated with influenza is vaccination. Vaccination is especially recommended for people in high-risk groups, such as residents of nursing or residential homes, as well as for diabetes, chronic renal failure, or chronic respiratory conditions.

Traditional methods of producing influenza vaccines involve growth of an isolated strain in embryonated hens' eggs. Initially, the virus is recovered from a throat swab or similar source and isolated in eggs. The initial isolation in egg is difficult, but the virus adapts to its egg host and subsequent propagation in eggs takes place relatively easily. It is widely recognized, however, that the egg-derived production of IV for vaccine purposes has several disadvantages. One disadvantage is that such production process is rather vulnerable due to the varying (micro)biological quality of the eggs. Another disadvantage is that the process completely lacks flexibility if demand suddenly increases, i.e., in case of a serious epidemic or pandemic, because of the logistical problems due to the non-availability of large quantities of suitable eggs. Also, vaccines thus produced are contra-indicated for persons with a known hypersensitivity to chicken and/or egg proteins.

The influenza vaccines currently in use are designated whole virus (WV) vaccine or subvirion (SV) (also called “split” or “purified surface antigen”). The WV vaccine contains intact, inactivated virus, whereas the SV vaccine contains purified virus disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus. Attenuated viral vaccines against influenza are also in development. A discussion of methods of preparing conventional vaccine may be found in Wright, P. F. & Webster, R. G., FIELDS VIROLOGY, 4d Ed. (Knipe, D. M. et al. Ed.), 1464-65 (2001), for example.

Virus Structures

An IV is roughly spherical, but it can also be elongated or irregularly shaped. Inside the virus, eight segments of single-stranded RNA contain the genetic instructions for making the virus. The most striking feature of the virus is a layer of spikes projecting outward over its surface. There are two different types of spikes: one is composed of the molecule hemagglutinin (HA), the other of neuraminidase (NA). The HA molecule allows the virus to “stick” to a cell, initiating infection. The NA molecule allows newly formed viruses to exit their host cell without sticking to the cell surface or to each other. The viral capsid is comprised of viral ribonucleic acid and several so called “internal” proteins (polymerases (PB1, PB2, and PA), matrix protein (M1) and nucleoprotein (NP)). Because antibodies against HA and NA have traditionally proved the most effective in fighting infection, much research has focused on the structure, function, and genetic variation of those molecules. Researchers are also interested in a two non-structural proteins M2 and NS1; both molecules play important roles in viral infection.

Type A subtypes are described by a nomenclature system that includes the geographic site of discovery; a lab identification number, the year of discovery, and in parentheses the type of HA and NA it possesses, for example, A/Hong Kong/156/97 (H5N1). If the virus infects non-humans, the host species is included before the geographical site, as in A/Chicken/Hong Kong/G9/97 (H9N2).

Virions contain 7 segments (influenza C virus) to 8 segments (influenza A and B virus) of linear negative-sense single stranded RNA. Most of the segments of the virus genome code for a single protein. For many influenza viruses, the whole genome is now known. Genetic reassortment of the virus results from intermixing of the parental gene segments in the progeny of the viruses when a cell is co-infected by two different viruses of a given type. This phenomenon is facilitated by the segmental nature of the genome of influenza virus. Genetic reassortment is manifested as sudden changes in the viral surface antigens.

Antigenic changes in HA and NA allow the influenza virus to have tremendous variability. Antigenic drift is the term used to indicate minor antigenic variations in HA and NA of the influenza virus from the original parent virus, while major changes in HA and NA which make the new virions significantly different, are called Antigenic shift. The difference between the two phenomena is a matter of degree.

Antigenic drift (minor changes) occurs due to accumulation of point mutations in the gene which results in changes in the amino acids in the proteins. Changes which are extreme, and drastic (too drastic to be explained by mutation alone) result in antigenic shift of the virus. The segmented genomes of the influenza viruses reassort readily in double infected cells. Genetic reassortment between human and non-human influenza virus has been suggested as a mechanism for antigenic shift. Influenza is a zoonotic disease, and an important pathogen in a number of animal species, including swine, horses, and birds, both wild and domestic. Influenza viruses are transferred to humans from other species.

Because of antigenic shift and antigenic drift, immunity to an IV carrying a particular HA and/or NA protein does not necessarily confer protective immunity against IV strains carrying variant, or different HA and/or NA proteins. Because antibodies against HA and NA have traditionally proved the most effective in fighting IV infection, much research has focused on the structure, function and genetic variation of those molecules.

Recent IV Vaccine Candidates

During the past few years, there has been substantial interest in testing DNA-based vaccines for a number of infectious diseases where the need for a vaccine, or an improved vaccine, exists. Several well-recognized advantages of DNA-based vaccines include the speed, ease and cost of manufacture, the versatility of developing and testing multivalent vaccines, the finding that DNA vaccines can produce a robust cellular response in a wide variety of animal models as well as in humans, and the proven safety of using plasmid DNA as a delivery vector (Donnelly, J. J., et al., Annu. Rev. Immunol. 15:617-648 (1997); Manickan, E., et al., Crit. Rev. Immunol. 17(2):139-154 (1997); U.S. Pat. No. 6,214,804). DNA vaccines represent the next generation in the development of vaccines (Nossal, G., Nat. Med. 4(5 Supple):475-476 (1998)) and numerous DNA vaccines are in clinical trials. The above references are herein incorporated by reference in their entireties.

Studies have already been performed using DNA-based vaccines in animals. Ulmer, J. B. et al., Science 259:1745-9 (1993) revealed that mice could be protected by an IV nucleoprotein DNA vaccine alone against severe disease and death resulting from either a homologous or a heterologous IV challenge. Further studies have substantiated this model, and comparative studies of live influenza vaccines versus DNA influenza vaccines show them to be relatively equivalent in immune induction and protection in the murine model.

WO 94/21797, incorporated herein by reference in its entirety, discloses IV vaccine compositions comprising DNA constructs encoding NP, HA, M1, PB1 and NS1. WO 94/21797 also discloses methods of protecting against IV infection comprising immunization with a prophylactically effective amount of these DNA vaccine compositions.

The IV nucleoprotein is relatively conserved (see Shu, L. L. et al., J. Virol. 67:2723-9 (1993)), but just as conserved are the M1 matrix protein (which is a major T-cell target), and the M2 protein, which are encoded by separate reading frames of RNA segment 7. See Neirynek, S. et al., Nat. Med. 5:1157-63 (1999); Lamb, R. A. & Lai, C. J., Virology 112:746-51 (1981); Ito, T. et al., J. Virol. 65:5491-8 (1991). Animal DNA vaccine trials have been performed with DNA constructs encoding these genes alone or in combination, usually with success. See Okuda, K., et al., Vaccine 19:3681-91 (2001); Watabe, S. et al., Vaccine 19:4434-44 (2001). Of interest, the M2 protein is involved as part of an ion channel, is critical in resistance to the antiviral agents amantadine and rimantadine, and approximately 24 amino acids are extracellular (eM2). See Fischer, W. B., Biochim Biophys Acta 1561:27-45 (2002); Zhong, Q., FEBS Lett 434:265-71 (1998). Antibodies to this extracellular, highly conserved protein (eM2), which is highly expressed in infected cells (Lamb, R. A., et al., Cell 40:627-33 (1985)), have been shown to be involved in animal models. Treanor, J. J., J. Virol. 64:1375-7 (1990); Slepushkin, V. A. et al., Vaccine 13:1399-402 (1995). An approach using a conjugate hepatitis B core-eM2 protein has been evaluated in an animal model and proposed as a pandemic influenza vaccine. Neirynck, S. et al., Nat. Med. 5:1157-63 (1999). However, in one study vaccination of pigs with a DNA construct expressing eM2-NP fusion protein exacerbated disease after challenge with influenza A virus. Heinen, P. P., J. Gen. Virol. 83:1851-59 (2002). All of the above references are herein incorporated by reference in their entireties

Heterologous “prime boost” strategies have been effective for enhancing immune responses and protection against numerous pathogens. Schneider et al., Immunol. Rev. 170:29-38 (1999); Robinson, H. L., Nat. Rev. Immunol. 2:239-50 (2002); Gonzalo, R. M. et al., Vaccine 20:1226-31 (2002); Tanghe, A., Infect. Immun. 69:3041-7 (2001). Providing antigen in different forms in the prime and the boost injections appears to maximize the immune response to the antigen. DNA vaccine priming followed by boosting with protein in adjuvant or by viral vector delivery of DNA encoding antigen appears to be the most effective way of improving antigen specific antibody and CD4+ T-cell responses or CD8+ T-cell responses respectively. Shiver J. W. et al., Nature 415: 331-5 (2002); Gilbert, S. C. et al., Vaccine 20:1039-45 (2002); Billaut-Mulot, O. et al., Vaccine 19:95-102 (2000); Sin, J. I. et al., DNA Cell Biol. 18:771-9 (1999). Recent data from monkey vaccination studies suggests that adding CRL1005 poloxamer (12 kDa, 5% POE), to DNA encoding the HIV gag antigen enhances T-cell responses when monkeys are vaccinated with an HIV gag DNA prime followed by a boost with an adenoviral vector expressing HIV gag (Ad5-gag). The cellular immune responses for a DNA/poloxamer prime followed by an Ad5-gag boost were greater than the responses induced with a DNA (without poloxamer) prime followed by Ad5-gag boost or for Ad5-gag only. Shiver, J. W. et al. Nature 415:331-5 (2002). U.S. Patent Appl. Publication No. US 2002/0165172 A1 describes simultaneous administration of a vector construct encoding an immunogenic portion of an antigen and a protein comprising the immunogenic portion of an antigen such that an immune response is generated. The document is limited to hepatitis B antigens and HIV antigens. Moreover, U.S. Pat. No. 6,500,432 is directed to methods of enhancing an immune response of nucleic acid vaccination by simultaneous administration of a polynucleotide and polypeptide of interest. According to the patent, simultaneous administration means administration of the polynucleotide and the polypeptide during the same immune response, preferably within 0-10 or 3-7 days of each other. The antigens contemplated by the patent include, among others, those of Hepatitis (all forms), HSV, HIV, CMV, EBV, RSV, VZV, HPV, polio, influenza, parasites (e.g., from the genus Plasmodium), and pathogenic bacteria (including but not limited to M. tuberculosis, M. leprae, Chlamydia, Shigella, B. burgdorferi, enterotoxigenic E. coli, S. typhosa, H. pylori, V. cholerae, B. pertussis, etc.). All of the above references are herein incorporated by reference in their entireties.

SUMMARY OF THE INVENTION

The present invention is directed to enhancing the immune response of a vertebrate in need of protection against IV infection by administering in vivo, into a tissue of the vertebrate, at least one polynucleotide, wherein the polynucleotide comprises one or more nucleic acid fragments, where the one or more nucleic acid fragments are optionally fragments of codon-optimized coding regions operably encoding one or more IV polypeptides, or fragments, variants, or derivatives thereof. The present invention is further directed to enhancing the immune response of a vertebrate in need of protection against IV infection by administering, in vivo, into a tissue of the vertebrate, a polynucleotide described above plus at least one isolated IV polypeptide or a fragment, a variant, or derivative thereof. The isolated IV polypeptide can be, for example, a purified subunit, a recombinant protein, a viral vector expressing an isolated IV polypeptide, or can be an inactivated or attentuated IV, such as those present in conventional IV vaccines. According to either method, the polynucleotide is incorporated into the cells of the vertebrate in vivo, and an immunologically effective amount of an immunogenic epitope of the encoded IV polypeptide, or a fragment, variant, or derivative thereof, is produced in vivo. When utilized, an isolated IV polypeptide or a fragment, variant, or derivative thereof is also administered in an immunologically effective amount.

According to the present invention, the polynucleotide can be administered either prior to, at the same time (simultaneously), or subsequent to the administration of the isolated IV polypeptide. The IV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide comprises at least one immunogenic epitope capable of eliciting an immune response to influenza virus in a vertebrate. In addition, an isolated IV polypeptide or fragment, variant, or derivative thereof, when used, comprises at least one immunogenic epitope capable of eliciting an immune response in a vertebrate. The IV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide can, but need not, be the same protein or fragment, variant, or derivative thereof as the isolated IV polypeptide which can be administered according to the method.

The polynucleotide of the invention can comprise a nucleic acid fragment, where the nucleic acid fragment is a fragment of a codon-optimized coding region operably encoding any IV polypeptide or fragment, variant, or derivative thereof, including, but not limited to, HA, NA, NP, M1 or M2 proteins or fragments (e.g., eM2), variants or derivatives thereof. A polynucleotide of the invention can also encode a derivative fusion protein, wherein two or more nucleic acid fragments, at least one of which encodes an IV polypeptide or fragment, variant, or derivative thereof, are joined in frame to encode a single polypeptide, e.g., NP fused to eM2. Additionally, a polynucleotide of the invention can further comprise a heterologous nucleic acid or nucleic acid fragment. Such heterologous nucleic acid or nucleic acid fragment may encode a heterologous polypeptide fused in frame with the polynucleotide encoding the IV polypeptide, e.g., a hepatitis B core protein or a secretory signal peptide. Preferably, the polynucleotide encodes an IV polypeptide or fragment, variant, or derivative thereof comprising at least one immunogenic epitope of IV, wherein the epitope elicits a B-cell (antibody) response, a T-cell (e.g., CTL) response, or both.

Similarly, the isolated IV polypeptide or fragment, variant, or derivative thereof to be delivered (either a recombinant protein, a purified subunit, or viral vector expressing an isolated IV polypeptide, or in the form of an inactivated IV vaccine) can be any isolated IV polypeptide or fragment, variant, or derivative thereof, including but not limited to the HA, NA, NP, M1 or M2 proteins or fragments (e.g., eM2), variants or derivatives thereof. In certain embodiments, a derivative protein can be a fusion protein, e.g., NP-eM2. In other embodiments, the isolated IV polypeptide or fragment, variant, or derivative thereof can be fused to a heterologous protein, e.g., a secretory signal peptide or the hepatitis B virus core protein. Preferably, the isolated IV polypeptide or fragment, variant, or derivative thereof comprises at least one immunogenic epitope of IV, wherein the antigen elicits a B-cell antibody response, a T-cell antibody response, or both.

Nucleic acids and fragments thereof of the present invention can be altered from their native state in one or more of the following ways. First, a nucleic acid or fragment thereof which encodes an IV polypeptide or fragment, variant, or derivative thereof can be part or all of a codon-optimized coding region, optimized according to codon usage in the animal in which the vaccine is to be delivered. In addition, a nucleic acid or fragment thereof which encodes an IV polypeptide can be a fragment which encodes only a portion of a full-length polypeptide, and/or can be mutated so as to, for example, remove from the encoded polypeptide non-desired protein motifs present in the encoded polypeptide or virulence factors associated with the encoded polypeptide. For example, the nucleic acid sequence could be mutated so as not to encode a membrane anchoring region that would prevent release of the polypeptide from the cell as with, e.g., eM2. Upon delivery, the polynucleotide of the invention is incorporated into the cells of the vertebrate in vivo, and a prophylactically or therapeutically effective amount of an immunologic epitope of an IV is produced in vivo.

Similarly, the proteins of the invention can be a fragment of a full-length IV polypeptide and/or can be altered so as to, for example, remove from the polypeptide non-desired protein motifs present in the polypeptide or virulence factors associated with the polypeptide. For example, the polypeptide could be altered so as not to encode a membrane anchoring region that would prevent release of the polypeptide from the cell.

The invention further provides immunogenic compositions comprising at least one polynucleotide, wherein the polynucleotide comprises one or more nucleic acid fragments, where each nucleic acid fragment is a fragment of a codon-optimized coding region encoding an IV polypeptide or a fragment, a variant, or a derivative thereof; and immunogenic compositions comprising a polynucleotide as described above and at least one isolated IV polypeptide or a fragment, a variant, or derivative thereof. Such compositions can further comprise, for example, carriers, excipients, transfection facilitating agents, and/or adjuvants as described herein.

The immunogenic compositions comprising a polynucleotide and an isolated IV polypeptide or fragment, variant, or derivative thereof as described above can be provided so that the polynucleotide and protein formulation are administered separately, for example, when the polynucleotide portion of the composition is administered prior (or subsequent) to the isolated IV polypeptide portion of the composition. Alternatively, immunogenic compositions comprising the polynucleotide and the isolated IV polypeptide or fragment, variant, or derivative thereof can be provided as a single formulation, comprising both the polynucleotide and the protein, for example, when the polynucleotide and the protein are administered simultaneously. In another alternative, the polynucleotide portion of the composition and the isolated IV polypeptide portion of the composition can be provided simultaneously, but in separate formulations.

Compositions comprising at least one polynucleotide comprising one or more nucleic acid fragments, where each nucleic acid fragment is optionally a fragment of a codon-optimized coding region operably encoding an IV polypeptide or fragment, variant, or derivative thereof together with and one or more isolated IV polypeptides or fragments, variants or derivatives thereof (as either a recombinant protein, a purified subunit, a viral vector expressing the protein, or in the form of an inactivated or attenuated IV vaccine) will be referred to herein as “combinatorial polynucleotide (e.g., DNA) vaccine compositions” or “single formulation heterologous prime-boost vaccine compositions.”

The compositions of the invention can be univalent, bivalent, trivalent or mulitvalent. A univalent composition will comprise only one polynucleotide comprising a nucleic acid fragment, where the nucleic acid fragment is optionally a fragment of a codon-optimized coding region encoding an IV polypeptide or a fragment, variant, or derivative thereof, and optionally the same IV polypeptide or a fragment, variant, or derivative thereof in isolated form. In a single formulation heterologous prime-boost vaccine composition, a univalent composition can include a polynucleotide comprising a nucleic acid fragment, where the nucleic acid fragment is optionally a fragment of a codon-optimized coding region encoding an IV polypeptide or a fragment, variant, or derivative thereof and an isolated polypeptide having the same antigenic region as the polynucleotide. A bivalent composition will comprise, either in polynucleotide or protein form, two different IV polypeptides or fragments, variants, or derivatives thereof, each capable of eliciting an immune response. The polynucleotide(s) of the composition can encode two IV polypeptides or alternatively, the polynucleotide can encode only one IV polypeptide and the second IV polypeptide would be provided by an isolated IV polypeptide of the invention as in, for example, a single formulation heterologous prime-boost vaccine composition. In the case where both IV polypeptides of a bivalent composition are delivered in polynucleotide form, the nucleic acid fragments operably encoding those IV polypeptides need not be on the same polynucleotide, but can be on two different polynucleotides. A trivalent or further multivalent composition will comprise three IV polypeptides or fragments, variants or derivatives thereof, either in isolated form or encoded by one or more polynucleotides of the invention.

The present invention further provides plasmids and other polynucleotide constructs for delivery of nucleic acid fragments of the invention to a vertebrate, e.g., a human, which provide expression of IV polypeptides, or fragments, variants, or derivatives thereof. The present invention further provides carriers, excipients, transfection-facilitating agents, immunogenicity-enhancing agents, e.g., adjuvants, or other agent or agents to enhance the transfection, expression or efficacy of the administered gene and its gene product.

In one embodiment, a mulitvalent composition comprises a single polynucleotide, e.g., plasmid, comprising one or more nucleic acid regions operably encoding IV polypeptides or fragments, variants, or derivatives thereof. Reducing the number of polynucleotides, e.g., plasmids in the compositions of the invention can have significant impacts on the manufacture and release of product, thereby reducing the costs associated with manufacturing the compositions. There are a number of approaches to include more than one expressed antigen coding sequence on a single plasmid. These include, for example, the use of Internal Ribosome Entry Site (IRES) sequences, dual promoters/expression cassettes, and fusion proteins.

The invention also provides methods for enhancing the immune response of a vertebrate to IV infection by administering to the tissues of a vertebrate one or more polynucleotides each comprising one or more nucleic acid fragments, where each nucleic acid fragment is optionally a fragment of a codon-optimized coding region encoding an IV polypeptide or fragment, variant, or derivative thereof; and optionally administering to the tissues of the vertebrate one or more isolated IV polypeptides, or fragments, variants, or derivatives thereof. The isolated IV polypeptide can be administered prior to, at the same time (simultaneously), or subsequent to administration of the polynucleotides encoding IV polypeptides.

In addition, the invention provides consensus amino acid sequences for IV polypeptides, or fragments, variants or derivatives thereof, including, but not limited to the HA, NA, NP, M1 or M2 proteins or fragments (e.g. eM2), variants or derivatives thereof. Polynucleotides which encode the consensus polypeptides or fragments, variants or derivatives thereof, are also embodied in this invention. Such polynucleotides can be obtained by known methods, for example by backtranslation of the amino acid sequence and PCR synthesis of the corresponding polynucleotide as described below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show an alignment of nucleotides 46-1542 of SEQ ID NO:1 (native NP coding region) with a coding region fully codon-optimized for human usage (SEQ ID NO:23).

FIG. 2 shows the protocol for the preparation of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005 and 5 mg/mil of DNA in a final volume of 3.6 ml, through the use of thermal cycling.

FIG. 3 shows the protocol for the preparation of a formulation comprising 0.3 mM BAK, 34 mg/ml or 50 mg/ml CRL 1005 and 2.5 mg/ml DNA in a final volume of 4.0 ml, through the use of thermal cycling.

FIG. 4 shows the protocol for the simplified preparation (without thermal cycling) of a formulation comprising 0.3 mM BAK, 7.5 mg/ml CRL 1005 and 5 mg/ml DNA.

FIG. 5 shows the anti-NP antibody response three weeks after a single administration of a combinatorial prime-boost vaccine formulation against the influenza virus NP protein.

FIG. 6 shows the anti-NP antibody response twelve days after a second administration of a combinatorial prime-boost vaccine formulation against the influenza virus NP protein.

FIG. 7 shows the CD8+ T Cell response to a combinatorial prime-boost vaccine formulation against the influenza virus NP protein.

FIG. 8 shows the CD4+ T Cell response to a combinatorial prime-boost vaccine formulation against the influenza virus NP protein.

FIGS. 9A and 9B show the results of a two dose mouse immunization regimen study with plasmid DNA encoding IAV HA (H3).

FIGS. 10A and 10B show the in vitro expression of M1 and M2 from segment 7 and an M1M2 fusion.

FIGS. 11A and 11B show the in vitro expression of eM2-NP and codon-optimized influenza virus NP protein.

FIGS. 12A-12D show the influenza A NP protein consensus amino acid sequence (SEQ ID NO: 76) aligned with 22 full length NP sequences. A dotted line indicates the same amino acid and a dashed line indicates that no sequence was available. Twenty-two NP full-length, or nearly full-length sequences were available for comparison on the World Wide Web at URL flu.lan1.gov. The amino acid chosen for the consensus sequences was based on the majority of the 22 sequences examined. In instances of a tie, the amino acid found in strain 2000 was favored.

FIG. 13 is a schematic diagram of various vectors encoding influenza proteins described herein.

FIG. 14 are the results of western blot experiments as described in Example 13, Experiment 3. The blots show lysates of VM92 cells transfected with plasmids which express M2 or NP to compare expression of the influenza protein from different expression vectors.

FIG. 15 are the results of western blot experiments as described in Example 13, Experiment 3. The blots show lysates of VM92 cells transfected with plasmids which express M1, M2 or NP to compare expression of the influenza protein from expression vectors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods for enhancing the immune response of a vertebrate in need of protection against IV infection by administering in vivo, into a tissue of a vertebrate, at least one polynucleotide comprising one or more nucleic acid fragments, where each nucleic acid fragment is optionally a fragment of a codon-optimized coding region operably encoding an IV polypeptide, or a fragment, variant, or derivative thereof in cells of the vertebrate in need of protection. The present invention is also directed to administering in vivo, into a tissue of the vertebrate the above described polynucleotide and at least one isolated IV polypeptide, or a fragment, variant, or derivative thereof. The isolated IV polypeptide or fragment, variant, or derivative thereof can be, for example, a recombinant protein, a purified subunit protein, a protein expressed and carried by a heterologous live or inactivated or attentuated viral vector expressing the protein, or can be an inactivated IV, such as those present in conventional, commercially available, inactivated IV vaccines. According to either method, the polynucleotide is incorporated into the cells of the vertebrate in vivo, and an immunologically effective amount of the influenza protein, or fragment or variant encoded by the polynucleotide is produced in vivo. The isolated protein or fragment, variant, or derivative thereof is also administered in an immunologically effective amount. The polynucleotide can be administered to the vertebrate in need thereof either prior to, at the same time (simultaneously), or subsequent to the administration of the isolated IV polypeptide or fragment, variant, or derivative thereof.

Non-limiting examples of IV polypeptides within the scope of the invention include, but are not limited to, NP, HA, NA, M1 and M2 polypeptides, and fragments, e.g., eM2, derivatives, e.g., an NP-eM2 fusion, and variants thereof. Nucleotide and amino acid sequences of IV polypeptides from a wide variety of IV types and subtypes are known in the art. The nucleotide sequences set out below are the wild-type sequences. For example, the nucleotide sequence of the NP protein of Influenza A/PR/8/34 (H1N1) is available as GenBank Accession Number M38279.1, and has the following sequence, referred to herein as SEQ ID NO:1:

AGCAAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAAATCATGGC
GTCTCAAGGCACCAAACGATCTTACGAACAGATGGAGACTGATGGAGAAC
GCCAGAATGCCACTGAAATCAGAGCATCCGTCGGAAAAATGATTGGTGGA
ATTGGACGATTCTACATCCAAATGTGCACCGAACTCAAACTCAGTGATTA
TGAGGGACGGTTGATCCAAAACAGCTTAACAATAGAGAGAATGGTGCTCT
CTGCTTTTGACGAAAGGAGAAATAAATACCTTGAAGAACATCCCAGTGCG
GGGAAAGATCCTAAGAAAACTGGAGGACCTATATACAGGAGAGTAAACGG
AAAGTGGATGAGAGAACTCATCCTTTATGACAAAGAAGAAATAAGGCGAA
TCTGGCGCCAAGCTAATAATGGTGACGATGCAACGGCTGGTCTGACTCAC
ATGATGATCTGGCATTCCAATTTGAATGATGCAACTTATCAGAGGACAAG
AGCTCTTGTTCGCACCGGAATGGATCCCAGGATGTGCTCTCTGATGCAAG
GTTCAACTCTCCCTAGGAGGTCTGGAGCCGCAGGTGCTGCAGTCAAAGGA
GTTGGAACAATGGTGATGGAATTGGTCAGAATGATCAAACGTGGGATCAA
TGATCGGAACTTCTGGAGGGGTGAGAATGGACGAAAAACAAGAATTGCTT
ATGAAAGAATGTGCAACATTCTCAAAGGGAAATTTCAAACTGCTGCACAA
AAAGCAATGATGGATCAAGTGAGAGAGAGCCGGAACCCAGGGAATGCTGA
GTTCGAAGATCTCACTTTTCTAGCACGGTCTGCACTCATATTGAGAGGGT
CGGTTGCTCACAAGTCCTGCCTGCCTGCCTGTGTGTATGGACCTGCCGTA
GCCAGTGGGTACGACTTTGAAAGGGAGGGATACTCTCTAGTCGGAATAGA
CCCTTTCAGACTGCTTCAAAACAGCCAAGTGTACAGCCTAATCAGACCAA
ATGAGAATCCAGCACACAAGAGTCAACTGGTGTGGATGGCATGCCATTCT
GCCGCATTTGAAGATCTAAGAGTATTAAGCTTCATCAAAGGGACGAAGGT
GCTCCCAAGAGGGAAGCTTTCCACTAGAGGAGTTCAAATTGCTTCCAATG
AAAATATGGAGACTATGGAATCAAGTACACTTGAACTGAGAAGCAGGTAC
TGGGCCATAAGGACCAGAAGTGGAGGAAACACCAATCAACAGAGGGCATC
TGCGGGCCAAATCAGCATACAACCTACGTTCTCAGTACAGAGAAATCTCC
CTTTTGACAGAACAACCGTTATGGCAGCATTCAGTGGGAATACAGAGGGG
AGAACATCTGACATGAGGACCGAAATCATAAGGATGATGGAAAGTGCAAG
ACCAGAAGATGTGTCTTTCCAGGGGCGGGGAGTCTTCGAGCTCTCGGACG
AAAAGGCAGCGAGCCCGATCGTGCCTTCCTTTGACATGAGTAATGAAGGA
TCTTATTTCTTCGGAGACAATGCAGAGGAATACGATAATTAAAGAAAAAT
ACCCTTGTTTCTACT

The amino acid sequence of the NP protein of Influenza A/PR/8/34 (H1N1), encoded by nucleotides 46-1494 of SEQ ID NO:1 is as follows, referred to herein as SEQ ID NO:2:

MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLS
DYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRV
NGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDATYQR
TRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRG
INDRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGN
AEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVG
IDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT
KVLPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQR
ASAGQISIQPTFSVQRNLPFDRTTVMAAFSGNTEGRTSDMRTEIIRMMES
ARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDN

Segment 7 of the IAV genome encodes both M1 and M2. Segment 7 of Influenza A virus (A/Puerto Rico/8/34/Mount Sinai (H1N1)), is available as GenBank Accession No. AF389121.1, and has the following sequence, referred to herein as SEQ ID NO:3:

AGCGAAAGCAGGTAGATATTGAAAGATGAGTCTTCTAACCGAGGTCGAAA
CGTACGTACTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCA
CAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACTGATCTTGAGGTTCT
CATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGA
TTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAG
CGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATCCAAATAA
CATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAACAT
TCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCC
AGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGT
GGCATTTGGCCTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGC
ATCGGTCTCATAGGCAAATGGTGACAACAACCAATCCACTAATCAGACAT
GAGAACAGAATGGTTTTAGCCAGCACTACAGCTAAGGCTATGGAGCAAAT
GGCTGGATCGAGTGAGCAAGCAGCAGAGGCCATGGAGGTTGCTAGTCAGG
CTAGACAAATGGTGCAAGCGATGAGAACCATTGGGACTCATCCTAGCTCC
AGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGGCCTATCAGAA
ACGAATGGGGGTGCAGATGCAACGGTTCAAGTGATCCTCTCGCTATTGCC
GCAAATATCATTGGGATCTTGCACTTGACATTGTGGATTCTTGATCGTCT
TTTTTTCAAATGCATTTACCGTCGCTTTAAATACGGACTGAAAGGAGGGC
CTTCTACGGAAGGAGTGCCAAAGTCTATGAGGGAAGAATATCGAAAGGAA
CAGCAGAGTGCTGTGGATGCTGACGATGGTCATTTTGTCAGCATAGAGCT
GGAGTAAAAAACTACCTTGTTTCTACT

The amino acid sequence of the M1 protein of Influenza A/Puerto Rico/8/34/Mount Sinai(H1N1), encoded by nucleotides 26 to 784 of SEQ ID NO:3 is as follows, referred to herein as SEQ ID NO:4:

MSLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRP
ILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLY
RKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEEVAFGLVCA
TCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQA
AEAMEVASQARQMVQAMRTIGTHPSSSAGLKNDLLENLQAYQKRMGVQMQ
RFK

The amino acid sequence of the M2 protein of Influenza A/Puerto Rico/8/34/Mount Sinai (H1N1), encoded (in spliced form) by nucleotides 26 to 51 and 740 to 1007 of SEQ ID NO:3 is as follows, referred to herein as SEQ ID NO:5:

MSLLTEVETPIRNEWGCRCNGSSDPLAIAANIIGILHLTLWILDRLFFKC
IYRRFKYGLKGGPSTEGVPKSMREEYRKEQQSAVDADDGHFVSIELE

The Extracellular region of the M2 protein (eM2) corresponds to the first 24 amino acids of the N-terminal end of the protein, and is underlined above. See Fischer, W. B. et al., Biochim. Biophys. Acta. 1561:27-45 (2002); Zhong, Q. et al., FEBS Lett. 434:265-71 (1998).

A derivative of NP and eM2 described herein is encoded by a construct which encodes the first 24 amino acids of M2 and all or a portion of NP. The fusion constructs may be constructed with the eM2 sequences followed by the NP sequences, or with the NP sequences followed by the eM2 sequences. Exemplary fusion constructs using the NP and M2 sequences from Influenza A/PR/8134 (H1N1) are set out below. A sequence, using the original influenza virus nucleotide sequences, which encodes the first 24 amino acids of M2 fused at its 3′ end to a sequence which encodes NP in its entirety eM2-NP is referred to herein as SEQ ID NO:6:

1 ATGAGTCTTC TAACCGAGGT CGAAACGCCT ATCAGAAACG AATGGGGGTG CAGATGCAAC
61 GGTTCAAGTG ATATGGCGTC TCAAGGCACC AAACGATCTT ACGAACAGAT GGAGACTGAT
121 GGAGAACGCC AGAATGCCAC TGAAATCAGA GCATCCGTCG GAAAAATGAT TGGTGGAATT
181 GGACGATTCT ACATCCAAAT GTGCACCGAA CTCAAACTCA GTGATTATGA GGGACGGTTG
241 ATCCAAAACA GCTTAACAAT AGAGAGAATG GTGCTCTCTG CTTTTGACGA AAGGAGAAAT
301 AAATACCTTG AAGAACATCC CAGTGCGGGG AAAGATCCTA AGAAAACTGG AGGACCTATA
361 TACAGGAGAG TAAACGGAAA GTGGATGAGA GAACTCATCC TTTATGACAA AGAAGAAATA
421 AGGCGAATCT GGCGCCAAGC TAATAATGGT GACGATGCAA CGGCTGGTCT GACTCACATG
481 ATGATCTGGC ATTCCAATTT GAATGATGCA ACTTATCAGA GGACAAGAGC TCTTGTTCGC
541 ACCGGAATGG ATCCCAGGAT GTGCTCTCTG ATGCAAGGTT CAACTCTCCC TAGGAGGTCT
601 GGAGCCGCAG GTGCTGCAGT CAAAGGAGTT GGAACAATGG TGATGGAATT GGTCAGAATG
661 ATCAAACGTG GGATCAATGA TCGGAACTTC TGGAGGGGTG AGAATGGACG AAAAACAAGA
721 ATTGCTTATG AAAGAATGTG CAACATTCTC AAAGGGAAAT TTCAAACTGC TGCACAAAAA
781 GCAATGATGG ATCAAGTGAG AGAGAGCCGG AACCCAGGGA ATGCTGAGTT CGAAGATCTC
841 ACTTTTCTAG CACGGTCTGC ACTCATATTG AGAGGGTCGG TTGCTCACAA GTCCTGCCTG
901 CCTGCCTGTG TGTATGGACC TGCCGTAGCC AGTGGGTACG ACTTTGAAAG GGAGGGATAC
961 TCTCTAGTCG GAATAGACCC TTTCAGACTG CTTCAAAACA GCCAAGTGTA CAGCCTAATC
1021 AGACCAAATG AGAATCCAGC ACACAAGAGT CAACTGGTGT GGATGGCATG CCATTCTGCC
1081 GCATTTGAAG ATCTAAGAGT ATTAAGCTTC ATCAAAGGGA CGAAGGTGCT CCCAAGAGGG
1141 AAGCTTTCCA CTAGAGGAGT TCAAATTGCT TCCAATGAAA ATATGGAGAC TATGGAATCA
1201 AGTACACTTG AACTGAGAAG CAGGTACTGG GCCATAAGGA CCAGAAGTGG AGGAAACACC
1261 AATCAACAGA GGGCATCTGC GGGCCAAATC AGCATACAAC CTACGTTCTC AGTACAGAGA
1321 AATCTCCCTT TTGACAGAAC AACCGTTATG GCAGCATTCA GTGGGAATAC AGAGGGGAGA
1381 ACATCTGACA TGAGGACCGA AATCATAAGG ATGATGGAAA GTGCAAGACC AGAAGATGTG
1441 TCTTTCCAGG GGCGGGGAGT CTTCGAGCTC TCGGACGAAA AGGCAGCGAG CCCGATCGTG
1501 CCTTCCTTTG ACATGAGTAA TGAAGGATCT TATTTCTTCG GAGACAATGC AGAGGAATAC
1561 GATAAT

The amino acid sequence of the eM2-NP fusion protein of Influenza A/PR/8/34/(H1N1), encoded by nucleotides 1 to 1566 SEQ ID NO:6 is as follows, referred to herein as SEQ ID NO:7 (eM2 amino acid sequence underlined):

MSLLTEVETPIRNEWGCRCNGSSDMASQGTKRSYEQMETDGERQNATEIR
ASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRN
KYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNG
DDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRS
GAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNIL
KGKFQTAAQKAMMDQVRESRNPGNAEFEDLTFLARSALILRGSVAHKSCL
PACVYGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKS
QLVWMACHSAAFEDLRVLSFIKGTKVLPRGKLSTRGVQIASNENMETMES
STLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPFDRTTVM
AAFSGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIV
PSFDMSNEGSYFFGDNAEEYDN

A sequence, using the original influenza virus nucleotide sequences, which encodes NP in its entirety fused at its 3′ end to the first 24 amino acids of M2 fused to a sequence which encodes NP in its entirety is referred to herein as SEQ ID NO:8:

ATGGCGTCTCAAGGCACCAAACGATCTTACGAACAGATGGAGACTGATGG
AGAACGCCAGAATGCCACTGAAATCAGAGCATCCGTCGGAAAAATGATTG
GTGGAATTGGACGATTCTACATCCAAATGTGCACCGAACTCAAACTCAGT
GATTATGAGGGACGGTTGATCCAAAACAGCTTAACAATAGAGAGAATGGT
GCTCTCTGCTTTTGACGAAAGGAGAAATAAATACCTTGAAGAACATCCCA
GTGCGGGGAAAGATCCTAAGAAAACTGGAGGACCTATATACAGGAGAGTA
AACGGAAAGTGGATGAGAGAACTCATCCTTTATGACAAAGAAGAAATAAG
GCGAATCTGGCGCCAAGCTAATAATGGTGACGATGCAACGGCTGGTCTGA
CTCACATGATGATCTGGCATTCCAATTTGAATGATGCAACTTATCAGAGG
ACAAGAGCTCTTGTTCGCACCGGAATGGATCCCAGGATGTGCTCTCTGAT
GCAAGGTTCAACTCTCCCTAGGAGGTCTGGAGCCGCAGGTGCTGCAGTCA
AAGGAGTTGGAACAATGGTGATGGAATTGGTCAGAATGATCAAACGTGGG
ATCAATGATCGGAACTTCTGGAGGGGTGAGAATGGACGAAAAACAAGAAT
TGCTTATGAAAGAATGTGCAACATTCTCAAAGGGAAATTTCAAACTGCTG
CACAAAAAGCAATGATGGATCAAGTGAGAGAGAGCCGGAACCCAGGGAAT
GCTGAGTTCGAAGATCTCACTTTTCTAGCACGGTCTGCACTCATATTGAG
AGGGTCGGTTGCTCACAAGTCCTGCCTGCCTGCCTGTGTGTATGGACCTG
CCGTAGCCAGTGGGTACGACTTTGAAAGGGAGGGATACTCTCTAGTCGGA
ATAGACCCTTTCAGACTGCTTCAAAACAGCCAAGTGTACAGCCTAATCAG
ACCAAATGAGAATCCAGCACACAAGAGTCAACTGGTGTGGATGGCATGCC
ATTCTGCCGCATTTGAAGATCTAAGAGTATTAAGCTTCATCAAAGGGACG
AAGGTGCTCCCAAGAGGGAAGCTTTCCACTAGAGGAGTTCAAATTGCTTC
CAATGAAAATATGGAGACTATGGAATCAAGTACACTTGAACTGAGAAGCA
GGTACTGGGCCATAAGGACCAGAAGTGGAGGAAACACCAATCAACAGAGG
GCATCTGCGGGCCAAATCAGCATACAACCTACGTTCTCAGTACAGAGAAA
TCTCCCTTTTGACAGAACAACCGTTATGGCAGCATTCAGTGGGAATACAG
AGGGGAGAACATCTGACATGAGGACCGAAATCATAAGGATGATGGAAAGT
GCAAGACCAGAAGATGTGTCTTTCCAGGGGCGGGGAGTCTTCGAGCTCTC
GGACGAAAAGGCAGCGAGCCCGATCGTGCCTTCCTTTGACATGAGTAATG
AAGGATCTTATTTCTTCGGAGACAATGCAGAGGAATACGATAATATGAGT
CTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTGCAGATG
CAACGGTTCAAGTGAT

The amino acid sequence of the NP-eM2 fusion protein of Influenza A/PR/8/34/(H1N1), encoded by nucleotides 1 to 1566 of SEQ ID NO:8 is as follows, referred to herein as SEQ ID NO:9 (eM2 amino acid sequence underlined):

MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSD
YEGRLIQMSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNG
KWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDATYQRTR
ALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGIN
DRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGNAE
FEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVGID
PFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGTKV
LPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRAS
AGQISIQPTFSVQRNLPFDRTTVMAAFSGNTEGRTSDMRTEIIRMMESAR
PEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDNMSLL
TEVETPIRNEWGCRCNGSSD

The construction of functional fusion proteins often requires a linker sequence between the two fused fragments, in order to adopt an extended conformation to allow maximal flexibility. We used program LINKER (Chiquita J. Crasto C. J. and Feng, J. Protein Engineering 13:309-312 (2000), program publicly available at chutney.med.yale.edu/linker/linker.html (visited Apr. 16, 2003)), that can automatically generate a set of linker sequences, which are known to adopt extended conformations as determined by X-ray crystallography and NMR. Examples of suitable linkers to use in various eM2-NP or NP-eM2 fusion proteins are as follows:

	GYNTRA	(SEQ ID NO:10)
	FQMGET	(SEQ ID NO:11)
	FDRVKHLK	(SEQ ID NO:12)
	GRNTNGVIT	(SEQ ID NO:13)
	VNEKTIPDHD	(SEQ ID NO:14)

The nucleotide sequence of the NP protein of Influenza B/LEE/40 is available as GenBank Accession Number K01395, and has the following sequence, referred to herein as SEQ ID NO:15:

1 ATGTCCAACA TGGATATTGA CAGTATAAAT ACCGGAACAA TCGATAAAAC ACCAGAAGAA
61 CTGACTCCCG GAACCAGTGG GGCAACCAGA CCAATCATCA AGCCAGCAAC CCTTGCTCCG
121 CCAAGCAACA AACGAACCCG AAATCCATCT CCAGAAAGGA CAACCACAAG CAGTGAAACC
181 GATATCGGAA GGAAAATCCA AAAGAAACAA ACCCCAACAG AGATAAAGAA GAGCGTCTAC
241 AAAATGGTGG TAAAACTGGG TGAATTCTAC AACCAGATGA TGGTCAAAGC TGGACTTAAT
301 GATGACATGG AAAGGAATCT AATTCAAAAT GCACAAGCTG TGGAGAGAAT CCTATTGGCT
361 GCAACTGATG ACAAGAAAAC TGAATACCAA AAGAAAAGGA ATGCCAGAGA TGTCAAAGAA
421 GGGAAGGAAG AAATAGACCA CAACAAGACA GGAGGCACCT TTTATAAGAT GGTAAGAGAT
481 GATAAAACCA TCTACTTCAG CCCTATAAAA ATTACCTTTT TAAAAGAAGA GGTGAAAACA
541 ATGTACAAGA CCACCATGGG GAGTGATGGT TTCAGTGGAC TAAATCACAT TATGATTGGA
601 CATTCACAGA TGAACGATGT CTGTTTCCAA AGATCAAAGG GACTGAAAAG GGTTGGACTT
661 GACCCTTCAT TAATCAGTAC TTTTGCCGGA AGCACACTAC CCAGAAGATC AGGTACAACT
721 GGTGTTGCAA TCAAAGGAGG TGGAACTTTA GTGGATGAAG CCATCCGATT TATAGGAAGA
781 GCAATGGCAG ACAGAGGGCT ACTGAGAGAC ATCAAGGCCA AGACGGCCTA TGAAAAGATT
841 CTTCTGAATC TGAAAAACAA GTGCTCTGCG CCGCAACAAA AGGCTCTAGT TGATCAAGTG
901 ATCGGAAGTA GGAACCCAGG GATTGCAGAC ATAGAAGACC TAACTCTGCT TGCCAGAAGC
961 ATGGTAGTTG TCAGACCCTC TGTAGCGAGC AAAGTGGTGC TTCCCATAAG CATTTATGCT
1021 AAAATACCTC AACTAGGATT CAATACCGAA GAATACTCTA TGGTTGGGTA TGAAGCCATG
1081 GCTCTTTATA ATATGGCAAC ACCTGTTTCC ATATTAAGAA TGGGAGATGA CGCAAAAGAT
1141 AAATCTCAAC TATTCTTCAT GTCGTGCTTC GGAGCTGCCT ATGAAGATCT AAGAGTGTTA
1201 TCTGCACTAA CGGGCACCGA ATTTAAGCCT AGATCAGCAC TAAAATGCAA GGGTTTCCAT
1261 GTCCCGGCTA AGGAGCAAGT AGAAGGAATG GGGGCAGCTC TGATGTCCAT CAAGCTTCAG
1321 TTCTGGGCCC CAATGACCAG ATCTGGAGGG AATGAAGTAA GTGGAGAAGG AGGGTCTGGT
1381 CAAATAAGTT GCAGCCCTGT GTTTGCAGTA GAAAGACCTA TTGCTCTAAG CAAGCAAGCT
1441 GTAAGAAGAA TGCTGTCAAT GAACGTTGAA GGACGTGATG CAGATGTCAA AGGAAATCTA
1501 CTCAAAATGA TGAATGATTC AATGGCAAAG AAAACCAGTG GAAATGCTTT CATTGGGAAG
1561 AAAATGTTTC AAATATCAGA CAAAAACAAA GTCAATCCCA TTGAGATTCC AATTAAGCAG
1621 ACCATCCCCA ATTTCTTCTT TGGGAGGGAC ACAGCAGAGG ATTATGATGA CCTCGATTAT
1681 TAA

The amino acid sequence of the NP protein of IBV B/LEE/40, encoded by nucleotides 1-1680 of SEQ ID NO: 15 is as follows, referred to herein as SEQ ID

MSNMDIDSINTGTIDKTPEELTPGTSGATRPIIKPATLAPPSNKRTRNPS
PERTTTSSETDIGRKIQKKQTPTEIKKSVYKMVVKLGEFYNQMMVKAGLN
DDMERNLIQNAQAVERILLAATDDKKTEYQKKRNARDVKEGKEEIDHNKT
GGTFYKMVRDDKTIYFSPIKITFLKEEVKTMYKTTMGSDGFSGLNHIMIG
HSQMNDVCFQRSKGLKRVGLDPSLISTFAGSTLPRRSGTTGVAIKGGGTL
VDEAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNKCSAPQQKALVDQV
IGSRNPGIADIEDLTLLARSMVVVRPSVASKVVLPISIYAKIPQLGFNTE
EYSMVGYEAMALYNMATPVSILRMGDDAKDKSQLFFMSCFGAAYEDLRVL
SALTGTEFKPRSALKCKGFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGG
NEVSGEGGSGQISCSPVFAVERPIALSKQAVRRMLSMNVEGRDADVKGNL
LKMMNDSMAKKTSGNAFIGKKMFQISDKNKVNPIEIPIKQTIPNFFFGRD
TAEDYDDLDY

Non limiting examples of nucleotide sequences encoding the IAV hemagglutinin (HA) are as follows. It should be noted that HA sequences vary significantly between IV subtypes. Virtually any nucleotide sequence encoding an IV HA is suitable for the present invention. In fact, HA sequences included in vaccines and therapeutic formulations of the present invention (discussed in more detail below) might change from year to year depending on the prevalent strain or strains of IV.

The partial nucleotide sequence of the HA protein of IAV A/New_York/1/18(H1N1) is available as GenBank Accession Number AF116576, and has the following sequence, referred to herein as SEQ ID NO:17:

1 atggaggcaa gactactggt cttgttatgt gcatttgcag ctacaaatgc agacacaata
61 tgtataggct accatgcgaa taactcaacc gacactgttg acacagtact cgaaaagaat
121 gtgaccgtga cacactctgt taacctgctc gaagacagcc acaacggaaa actatgtaaa
181 ttaaaaggaa tagccccatt acaattgggg aaatgtaata tcgccggatg gctcttggga
241 aacccggaat gcgatttact gctcacagcg agctcatggt cctatattgt agaaacatcg
301 aactcagaga atggaacatg ttacccagga gatttcatcg actatgaaga actgagggag
361 caattgagct cagtgtcatc gtttgaaaaa ttcgaaatat ttcccaagac aagctcgtgg
421 cccaatcatg aaacaaccaa aggtgtaacg gcagcatgct cctatgcggg agcaagcagt
481 ttttacagaa atttgctgtg gctgacaaag aagggaagct catacccaaa gcttagcaag
541 tcctatgtga acaataaagg gaaagaagtc cttgtactat ggggtgttca tcatccgcct
601 accggtactg atcaacagag tctctatcag aatgcagatg cttatgtctc tgtagggtca
661 tcaaaatata acaggagatt caccccggaa atagcagcga gacccaaagt aagaggtcaa
721 gctgggagga tgaactatta ctggacatta ctagaacccg gagacacaat aacatttgag
781 gcaactggaa atctaatagc accatggtat gctttcgcac tgaatagagg ttctggatcc
841 ggtatcatca cttcagacgc accagtgcat gattgtaaca cgaagtgtca aacaccccat
901 ggtgctataa acagcagtct ccctttccag aatatacatc cagtcacaat aggagagtgc
961 ccaaaatacg tcaggagtac caaattgagg atggctacag gactaagaaa cattccatct
1021 attcaatcca ggggtctatt tggagccatt gccggtttta ttgagggggg atggactgga
1081 atgatagatg gatggtatgg ttatcatcat cagaatgaac agggatcagg ctatgcagcg
1141 gatcaaaaaa gcacacaaaa tgccattgac gggattacaa acaaggtgaa ttctgttatc
1201 gagaaaatga acacccaatt

The amino acid sequence of the partial HA protein of IAV A/New_York/1/18(H1N1), encoded by nucleotides 1 to 1218 of SEQ ID NO:17 is as follows, referred to herein as SEQ ID NO:18:

MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL

EDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETS

NSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNIIETTKGV

TAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHP

PTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARLPKVRGQAGRMNYYW

TLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQT

PHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFG

AIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNS

VIEKMNTQ

The nucleotide sequence of the IAV A/Hong Kong/482/97 hemagglutinin (H5) is available as GenBank Accession Number AF046098, and has the following sequence, referred to herein as SEQ ID NO:19:

1 ctgtcaaaat ggagaaaata gtgcttcttc ttgcaacagt cagtcttgtt aaaagtgatc
61 agatttgcat tggttaccat gcaaacaact cgacagagca ggttgacaca ataatggaaa
121 agaatgttac tgttacacat gcccaagaca tactggaaag gacacacaac gggaagctct
181 gcgatctaaa tggagtgaaa cctctcattt tgagggattg tagtgtagct ggatggctcc
241 tcggaaaccc tatgtgtgac gaattcatca atgtgccgga atggtcttac atagtggaga
301 aggccagtcc agccaatgac ctctgttatc cagggaattt caacgactat gaagaactga
361 aacacctatt gagcagaata aaccattttg agaaaattca gatcatcccc aaaagttctt
421 ggtccaatca tgatgcctca tcaggggtga gctcagcatg tccatacctt gggaggtcct
481 cctttttcag aaatgtggta tggcttatca aaaagaacag tgcataccca acaataaaga
541 ggagctacaa taataccaac caagaagatc ttttggtact gtgggggatt caccatccta
601 atgatgcggc agageagaca aagctctatc aaaatccaac cacctacatt tccgttggaa
661 catcaacact gaaccagaga ttggttccag aaatagctac tagacccaaa gtaaacgggc
721 aaagtggaag aatggagttc ttctggacaa ttttaaagcc gaatgatgcc atcaatttcg
781 agagtaatgg aaatttcatt gccccagaat atgcatacaa aattgtcaag aaaggggact
841 caacaattat gaaaagtgaa ttggaatatg gtaactgcaa caccaagtgt caaactccaa
901 tgggggcgat aaactctagt atgccattcc acaacataca ccccctcacc atcggggaat
961 gccccaaata tgtgaaatca aacagattag ttcttgcgac tggactcaga aatacccctc
1021 aaagggagag aagaagaaaa aagagaggac tatttggagc tatagcaggt tttatagagg
1081 gaggatggca gggcatggta gatggttggt atgggtacca ccatagcaat gagcagggga
1141 gtggatacgc tgcagacaaa gaatccactc aaaaggcaat agatggagtc accaataagg
1201 tcaactcgat cattaacaaa atgaacactc agtttgaggc cgttggaagg gaatttaata
1261 acttagaaag gagaatagag aatttaaaca agaaaatgga agacggattc ctagatgtct
1321 ggacttacaa tgctgaactt ctggttctca tggaaaatga gagaactctc gactttcatg
1381 actcaaatgt caagaacctt tacgacaagg tccgactaca gcttagggat aatgcaaagg
1441 aactgggtaa tggttgtttc gaattctatc acaaatgtga taatgaatgt atggaaagtg
1501 taaaaaacgg aacgtatgac tacccgcagt attcagaaga agcaagacta aacagagagg
1561 aaataagtgg agtaaaattg gaatcaatgg gaacttacca aatactgtca atttattcaa
1621 cagtggcgag ttccctagca ctggcaatca tggtagctgg tctatcttta tggatgtgct
1681 ccaatggatc gttacaatgc agaatttgca tttaaatttg tgagttcaga ttgtagttaa
1741 a

The amino acid sequence of the HA protein of IAV A/Hong Kong/482/97 (H5), encoded by nucleotides 9 to 1715 of SEQ ID NO:19 is as follows, referred to herein as SEQ ID NO:20:

MEKIVLLLATVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE

RTHNGKLCDLNGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAS

PANDLCYPGNFNDYEELKLHLLSRINHFEKIQIIPKSSWSNHDASSGVSS

ACPYLGRSSFFRNVVWLIKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPND

AAEQTKLYQNPTTYISVGTSTLNQRLVPEIATRPKVNGQSGRMEFFWTIL

KPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMG

AINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQRERRRKKRGLF

GAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVN

SIINKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME

NERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVK

NGTYDYPQYSEEARLNREEISGVKLESMGTYQILSIYSTVASSLALAIMV

AGLSLWMCSNGSLQCRICI

The nucleotide sequence of the IAV A/Hong Kong/1073/99(H9N2) is available as GenBank Accession Number INA404626, and has the following sequence, referred to herein as SEQ ID NO:21:

1 gcaaaagcag gggaattact taactagcaa aatggaaaca atatcactaa taactatact
61 actagtagta acagcaagca atgcagataa aatctgcatc ggccaccagt caacaaactc
121 cacagaaact gtggacacgc taacagaaac caatgttcct gtgacacatg ccaaagaatt
181 gctccacaca gagcataatg gaatgctgtg tgcaacaagc ctgggacatc ccctcattct
241 agacacatgc actattgaag gactagtcta tggcaaccct tcttgtgacc tgctgttggg
301 aggaagagaa tggtcctaca tcgtcgaaag atcatcagct gtaaatggaa cgtgttaccc
361 tgggaatgta gaaaacctag aggaactcag gacacttttt agttccgcta gttcctacca
421 aagaatccaa atcttcccag acacaacctg gaatgtgact tacactggaa caagcagagc
481 atgttcaggt tcattctaca ggagtatgag atggctgact caaaagagcg gtttttaccc
541 tgttcaagac gcccaataca caaataacag gggaaagagc attcttttcg tgtggggcat
601 acatcaccca cccacctata ccgagcaaac aaatttgtac ataagaaacg acacaacaac
661 aagcgtgaca acagaagatt tgaataggac cttcaaacca gtgatagggc caaggcccct
721 tgtcaatggt ctgcagggaa gaattgatta ttattggtcg gtactaaaac caggccaaac
781 attgcgagta cgatccaatg ggaatctaat tgctccatgg tatggacacg ttctttcagg
841 agggagccat ggaagaatcc tgaagactga tttaaaaggt ggtaattgtg tagtgcaatg
901 tcagactgaa aaaggtggct taaacagtac attgccattc cacaatatca gtaaatatgc
961 atttggaacc tgccccaaat atgtaagagt taatagtctc aaactggcag tcggtctgag
1021 gaacgtgcct gctagatcaa gtagaggact atttggagcc atagctggat tcatagaagg
1081 aggttggcca ggactagtcg ctggctggta tggtttccag cattcaaatg atcaaggggt
1141 tggtatggct gcagataggg attcaactca aaaggcaatt gataaaataa catccaaggt
1201 gaataatata gtcgacaaga tgaacaagca atatgaaata attgatcatg aattcagtga
1261 ggttgaaact agactcaata tgatcaataa taagattgat gaccaaatac aagacgtatg
1321 ggcatataat gcagaattgc tagtactact tgaaaatcaa aaaacactcg atgagcatga
1381 tgcgaacgtg aacaatctat ataacaaggt gaagagggca ctgggctcca atgctatgga
1441 agatgggaaa ggctgtttcg agctatacca taaatgtgat gatcagtgca tggaaacaat
1501 tcggaacggg acctataata ggagaaagta tagagaggaa tcaagactag aaaggcagaa
1561 aatagagggg gttaagctgg aatctgaggg aacttacaaa atcctcacca tttattcgac
1621 tgtcgcctca tctcttgtgc ttgcaatggg gtttgctgcc ttcctgttct gggccatgtc
1681 caatggatct tgcagatgca acatttgtat ataa

The amino acid sequence of the HA protein of IAV A/Hong Kong/1073/99 (H9N2), encoded by nucleotides 32 to 1711 of SEQ ID NO:21 is as follows, referred to herein as SEQ ID NO:22:

METISLITILLVVTASNADKICIGHQSTNSTETVDTLTETNVPVTHAKEL

LHTEHNGMLCATSLGHPLILDTCTIEGLVYGNPSCDLLLGGREWSYIVER

SSAVNGTCYPGNVENLEELRTLFSSASSYQRIQIFPDTTWNVTYTGTSRA

CSGSFYRSMRWLTQKSGFYPVQDAQYTNNRGKSILFVWGIHHPPTYTEQT

NLYIRNDTTTSVTTEDLNRTFKPVIGPRPLVNGLQGRIDYYWSVLKPGQT

LRVRSNGNLIAPWYGHVLSGGSHGRILKTDLKGGNCVVQCQTEKGGLNST

LPFHNISKYAFGTCPKYVRVNSLKLAVGLRNVPARSSRGLFGAIAGFIEG

GWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQ

YEIIDHEFSEVETRINMINNKIDDQIQDVWAYNAELLVLLENQKTLDEHD

ANVNNLYNKVKRALGSNAMEDGKGCFELYHKCDDQCMETIRNGTYNRRKY

REESRLERQKIEGVKLESEGTYKILTIYSTVASSLVLAMGFAAFLFWAMS

NGSCRCNICI

The present invention also provides vaccine compositions and methods for delivery of IV coding sequences to a vertebrate with optimal expression and safety conferred through codon optimization and/or other manipulations. These vaccine compositions are prepared and administered in such a manner that the encoded gene products are optimally expressed in the vertebrate of interest. As a result, these compositions and methods are useful in stimulating an immune response against IV infection. Also included in the invention are expression systems, delivery systems, and codon-optimized IV coding regions.

In a specific embodiment, the invention provides combinatorial polynucleotide (e.g., DNA) vaccines which combine both a polynucleotide vaccine and polypeptide (e.g., either a recombinant protein, a purified subunit protein, a viral vector expressing an isolated IV polypeptide, or in the form of an inactivated or attenuated IV vaccine) vaccine in a single formulation. The single formulation comprises an IV polypeptide-encoding polynucleotide vaccine as described herein, and optionally, an effective amount of a desired isolated IV polypeptide or fragment, variant, or derivative thereof. The polypeptide may exist in any form, for example, a recombinant protein, a purified subunit protein, a viral vector expressing an isolated IV polypeptide, or in the form of an inactivated or attenuated IV vaccine. The IV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide vaccine may be identical to the isolated IV polypeptide or fragment, variant, or derivative thereof. Alternatively, the IV polypeptide or fragment, variant, or derivative thereof encoded by the polynucleotide may be different from the isolated IV polypeptide or fragment, variant, or derivative thereof.

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

The term “polynucleotide” is intended to encompass a singular nucleic acid or nucleic acid fragment as well as plural nucleic acids or nucleic acid fragments, and refers to an isolated molecule or construct, e.g., a virus genome (e.g., a non-infectious viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles as described in (Darquet, A-M et al., Gene Therapy 4:1341-1349 (1997)) comprising a polynucleotide. A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

The terms “nucleic acid” or “nucleic acid fragment” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide or construct. A nucleic acid or fragment thereof may be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form as well as double-stranded or single-stranded forms. By “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.

As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, and the like, are not part of a coding region. Two or more nucleic acids or nucleic acid fragments of the present invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate polynucleotide constructs, e.g., on separate (different) plasmids. Furthermore, any nucleic acid or nucleic acid fragment may encode a single IV polypeptide or fragment, derivative, or variant thereof, e.g., or may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides. In addition, a nucleic acid may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator, or may encode heterologous coding regions fused to the IV coding region, e.g., specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

The terms “fragment,” “variant,” “derivative” and “analog” when referring to IV polypeptides of the present invention include any polypeptides which retain at least some of the immunogenicity or antigenicity of the corresponding native polypeptide. Fragments of IV polypeptides of the present invention include proteolytic fragments, deletion fragments and in particular, fragments of IV polypeptides which exhibit increased secretion from the cell or higher immunogenicity or reduced pathogenicity when delivered to an animal. Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three-dimensional epitopes. Variants of IV polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally, such as an allelic variant. By an “allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome or genome of an organism or virus. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985), which is incorporated herein by reference. For example, as used herein, variations in a given gene product is a “variant”. When referring to IV NA or HA proteins, each such protein is a “variant,” in that native IV strains are distinguished by the type of NA and HA proteins encoded by the virus. However, within a single HA or NA variant type, further naturally or non-naturally occurring variations such as amino acid deletions, insertions or substitutions may occur. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of IV polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. An analog is another form of an IV polypeptide of the present invention. An example is a proprotein which can be activated by cleavage of the proprotein to produce an active mature polypeptide.

The terms “infectious polynucleotide” or “infectious nucleic acid” are intended to encompass isolated viral polynucleotides and/or nucleic acids which are solely sufficient to mediate the synthesis of complete infectious virus particles upon uptake by permissive cells. Thus, “infectious nucleic acids” do not require pre-synthesized copies of any of the polypeptides it encodes, e.g., viral replicases, in order to initiate its replication cycle in a permissive host cell.

The terms “non-infectious polynucleotide” or “non-infectious nucleic acid” as defined herein are polynucleotides or nucleic acids which cannot, without additional added materials, e.g, polypeptides, mediate the synthesis of complete infectious virus particles upon uptake by permissive cells. An infectious polynucleotide or nucleic acid is not made “non-infectious” simply because it is taken up by a non-permissive cell. For example, an infectious viral polynucleotide from a virus with limited host range is infectious if it is capable of mediating the synthesis of complete infectious virus particles when taken up by cells derived from a permissive host (i.e., a host permissive for the virus itself). The fact that uptake by cells derived from a non-permissive host does not result in the synthesis of complete infectious virus particles does not make the nucleic acid “non-infectious.” In other words, the term is not qualified by the nature of the host cell, the tissue type, or the species taking up the polynucleotide or nucleic acid fragment.

In some cases, an isolated infectious polynucleotide or nucleic acid may produce fully-infectious virus particles in a host cell population which lacks receptors for the virus particles, i.e., is non-permissive for virus entry. Thus viruses produced will not infect surrounding cells. However, if the supernatant containing the virus particles is transferred to cells which are permissive for the virus, infection will take place.

The terms “replicating polynucleotide” or “replicating nucleic acid” are meant to encompass those polynucleotides and/or nucleic acids which, upon being taken up by a permissive host cell, are capable of producing multiple, e.g., one or more copies of the same polynucleotide or nucleic acid. Infectious polynucleotides and nucleic acids are a subset of replicating polynucleotides and nucleic acids; the terms are not synonymous. For example, a defective virus genome lacking the genes for virus coat proteins may replicate, e.g., produce multiple copies of itself, but is NOT infectious because it is incapable of mediating the synthesis of complete infectious virus particles unless the coat proteins, or another nucleic acid encoding the coat proteins, are exogenously provided.

In certain embodiments, the polynucleotide, nucleic acid, or nucleic acid fragment is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally also comprises a promoter and/or other transcription or translation control elements operably associated with the polypeptide-encoding nucleic acid fragment. An operable association is when a nucleic acid fragment encoding a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide-encoding nucleic acid fragment and a promoter associated with the 5′ end of the nucleic acid fragment) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid fragment encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid fragment. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, elements from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

A DNA polynucleotide of the present invention may be a circular or linearized plasmid or vector, or other linear DNA which may also be non-infectious and nonintegrating (i.e., does not integrate into the genome of vertebrate cells). A linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease. Linear DNA may be advantageous in certain situations as discussed, e.g., in Cherng, J. Y., et al.,

1. An isolated polypeptide produced by a polynucleotide comprising a nucleic acid fragment which encodes the amino acid sequence of SEQ ID NO:76, wherein the codons of said nucleic acid fragment are optimized for expression in humans.

2. The isolated polypeptide of claim 1, wherein said nucleic acid fragment encoding the isolated polypeptide comprises SEQ ID NO:75.

3. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:76, wherein said polypeptide, upon administration to a vertebrate elicits a detectable immune response against SEQ ID NO:76.

4. The polypeptide of claims 3, further comprising a heterologous polypeptide ligated to said polypeptide.

5. The polypeptide of claim 4, wherein said heterologous polypeptide is hepatitis B core antigen.

6. The polypeptide of claim 5, wherein said hepatitis core antigen comprises at least 50 amino acids of a polypeptide selected from the group consisting of SEQ ID NO:40 and SEQ ID NO:42.

7. The polypeptide of claim 3, wherein said heterologous polypeptide comprises at least the extracellular domain of an influenza virus M2 protein.

8. A composition comprising the polypeptide of claim 1 and an adjuvant.

9. A method of treating or preventing influenza infection in a vertebrate comprising administering to a vertebrate in need thereof the composition of claim 8.

10. A composition comprising the polypeptide of claim 3 and an adjuvant.

11. A method of treating or preventing influenza infection in a vertebrate comprising administering to a vertebrate in need thereof the composition of claim 10.