Plant-made west nile virus (WNV) vaccines, vectors and plant codon optimized sequences

This application claims the benefit of U.S. Provisional Application Ser. No. 60/871,518, filed Dec. 22, 2006, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

This invention was made with government support under USDA-ARS CRADA Agreement No. 58-3K95-M-1040. The government has certain rights in the invention.

Understanding of West Nile virus (WNV) neutralization by antibodies comes from the study of WNV and its close relatives Saint Louis encephalitis virus, Murray Valley encephalitis virus (MVEV), and Japanese encephalitis virus, as well as more distant relatives such as dengue virus, yellow fever virus, and tick-borne encephalitis virus (TBEV). Strong similarities in the sequence of the flavivirus envelope proteins and the nearly identical position of the cysteines that form intra-molecular bonds within the envelope proteins (Nowak et al., 1987) suggest that the envelope proteins of all flaviviruses must have very similar structures. Therefore, information about any flavivirus is generally applicable to the others.

Heinz and Kunz (1982, 1977) showed that flaviviruses contain only three proteins, the envelope protein (E), the membrane protein (M), and the capsid protein (C). Recent structural studies of dengue virus (Kuhn et al., 2002) confirmed this and showed the physical relationship of these proteins in the virion. Only the E protein is exposed on the virion surface (Kuhn et al., 2002). Thus far, the three-dimensional structures of the E proteins for WNV, TBEV, and dengue have been solved (Kuhn et al., 2002; Mukhopadhyah et al., 2003; Rey et al., 1995). These have a finger-like structure with three clearly distinct domains, domain I being in the middle between domains II and III. Individual molecules of E protein lay flat across the virion surface with pairs of molecules lying beside each other in opposite orientation and three pairs laying side-by-side.

The flavivirus E protein is synthesized as part of a genome-length polyprotein that includes all viral proteins. It is subsequently released from the polyprotein by proteolytic cleavage. Early cleavages inside the polyprotein release the E protein still attached to the pre-membrane (pr) and M proteins, the combination of the pr and M proteins being known as the “prM” protein. The resulting prM-E protein is inserted into the endoplasmic reticulum membrane where it begins to fold into its mature conformation. The virus is assembled in intracellular compartments with the prM-E on the surface. Subsequent cleavages separate the E and prM proteins and cleave the prM to yield the mature M protein. The pr fragment is not incorporated into virions. The E protein may or may not have glycosylation sequences and therefore may or may not be glycosylated (Hanna, et al., 2005).

Flaviviruses infect cells by binding to the cell membrane, probably through an interaction between the RGD sequence of E protein domain III and cell-surface integrin (Lee et al., 2000), and entering through endosomes. When the endosome acidifies, the virion envelope proteins undergo extensive and irreversible changes in their intra- and inter-molecular conformation. The 180 individual E protein molecules disassociate from their dimers, reorient their domains and join to form 60 trimeric spikes that protrude from the virion membrane, insert the tip of the spikes into the endosomal membrane, and aggregate into 12 pentameric rings of trimeric spikes that fuse the virion membrane with the endosomal membrane, thus allowing the capsid to enter the cell's cytoplasm and begin replication (Bressanelli et al., 2004). It is clear that solubilization of the dimers from the virion surface ablates some neutralization-related epitopes (Heinz et al., 1991) but it is not clear how the rearrangement and trimerization alters E protein antigenic sites (Stiasny et al., 1996).

Since only the E protein is exposed on the virion surface, antibodies that bind to and neutralize intact, infectious virions must bind to the E protein. This has been proven by showing the development of neutralizing antibodies in animals immunized with proteins purified from virus (Heinz et al., 1990) and viral proteins produced in recombinant systems (Bray et al., 1989; Heinz et al., 1986; Heinz et al., 1982; Jan et al., 1993; Konishi et al., 1992; Mason et al., 1991; Men et al., 1991; Pincus et al., 1992; Schlesinger et al., 1992), and by passive protection experiments with monoclonal antibodies directed against the E protein (reviewed in Heinz et al., 1977, 1986; Roehrig 1986).

Antibodies that bind some areas on the E protein would be expected to neutralize the virus and antibodies that bind other areas might not. In order to discriminate between the neutralization activity of antibodies that bind the primary amino acid sequence from those that bind the secondary and tertiary structure of the properly folded E protein, Wengler and Wengler (1989) showed that reduction of disulfide bonds to destroy the protein's secondary and tertiary structure ablated the ability of WNV E protein to engender neutralizing antibodies. This experiment strongly suggested that neutralizing antibodies bind to the E protein secondary and tertiary conformational structure rather than linear structure. To confirm this, Roehrig et al. (1989) made peptides from MVEV E protein predicted epitopes and found that only one engendered neutralizing antibodies and only at a low level. Indeed, subsequent studies have shown that monoclonal antibodies usually bind either native E protein or denatured E protein and its peptides (Guirakhoo et al., 1989; Holzmann et al., 1993; Roehrig et al., 1989). Only antibodies that bind the native structure neutralize the virus.

To show exactly which areas of the E protein are attacked by neutralizing antibodies, mutations in viruses that have escaped neutralization by monoclonal antibodies were sequenced and mapped on the E protein surface (reviewed in Heinz et al., 1983; Heinz et al., 1990; Roehrig 1986). These data enabled the generation of crude structural models (Cammack et al., 1986; Kolaskar et al., 1999; Mandl et al., 1989; Roebrig et al., 1989; Roehrig et al.; 1983) that were subsequently refined to show that mutations mapped to all three structural domains defined by x-ray crystallographic methods (Cecilia et al., 1991; Gao et al., 1994; Hasegawa et al., 1992; Holzmann et al., 1997; Holzmann et al., 1993; Jiang et al., 1993; Lin et al., 1994; Mandl et al., 1989). This strongly suggests that antibodies can neutralize flaviviruses by binding to any of the three domains. Nevertheless, most studies have focused on domain III where many neutralizing monoclonal antibody escape mutations occur (Beasley et al., 2002). Domain III is also the binding site for some non-neutralizing antibodies (Sanchez et al., 2005). Domain III can be isolated from purified virions as a trypsin-resistant fragment (Winkler et al., 1987) or generated as a recombinant protein (Mason et al. 1989) but its reactivity with neutralizing monoclonal antibodies is dependent on the maintenance of its conformational structure by its single disulfide bond. Several antibodies appear to neutralize WNV by binding a peptide that is exposed on domain I only during the membrane fusion transition (Kanai et al., 2006) or a site that interferes with conformational changes in domain III (Nybakken et al., 2005).

The subject application provides various compositions of matter directed to West Nile virus (WNV) polypeptides and fragments thereof and polynucleotides, vectors and transformed host cells that encode, direct the expression of, or produce WNV polypeptides as set forth herein. Methods of using the polypeptides and polynucleotides for the production of immune responses in individuals or detecting the presence of WNV specific or neutralizing antibodies are also provided herein.

FIG. 1 depicts plasmid pDAB2406 which contains the cassava vein mosaic virus (CsVMV) promoter described in WO 97/48819 and an open reading frame 3′ untranslated region, ORF23 3′UTR (GenBank accession number X00493) v1. Located between the CsVMV promoter and ORF23 3′UTR v1 are unique sites, NcoI and SacI, which were used for inserting the gene of interest.

FIG. 2 represents vector pDAB2418. pDAB2418 contains the RB7 matrix attachment region (MAR) (U.S. Pat. No. 5,773,689; U.S. Pat. No. 5,773,695; U.S. Pat. No. 6,239,328, WO 94/07902, and WO 97/27207) and the plant transcription unit where plant selection marker phosphinothricin acetyl transferase (PAT) (U.S. Pat. Nos. 5,879,903; 5,637,489; 5,276,268; and 5,273,894) is driven by the AtUbi10 promoter (Sun C.-W. et al., 1997; Norris, S. R. et al., 1993; Callis, J. et al, 1995) and flanked, downstream by AtuORF1 3′ UTR v3 (U.S. Pat. No. 5,428,147; Barker, R. F., et al., 1983; GenBank accession number X00493). A unique NotI site, located between the RB7 MAR gene and the plant AtUbi10 promoter, was used for cloning gene fragments from pDAB2406 containing the CsVMV promoter, gene of interest, and ORF23 3′UTR v1.

FIG. 3 illustrates a modified basic binary vector, pDAB2407. This binary vector was built by adding an AgeI linker at the unique BamHI site of pBBV (Basic Binary Vector) allowing for AgeI/AgeI ligation of the WNV antigen and selectable marker expression cassettes between the T-DNA borders.

FIG. 4 is a representation of West Nile Virus dicot binary vector pDAB2475 which encodes a chimeric protein consisting of tobacco codon biased West Nile Virus membrane and envelope peptide (version 2) with ER targeting v2 and KDEL retention v3 signals (SEQ ID NO: 12).

FIG. 5 depicts a dicot binary vector (pDAB2478) encoding a chimeric protein consisting of the tobacco codon biased West Nile Virus pre-membrane v2, membrane and envelope peptides v2 with ER targeting v2 and KDEL retention v3 signals (SEQ ID NO: 8).

FIG. 6 pertains to a dicot binary vector, pDAB2481, encoding a chimeric protein consisting of the tobacco codon biased West Nile Virus pre-membrane v2, membrane v2, and envelope peptides with a mutated N-glycosylation site (version 4) with ER targeting v2 and KDEL v3 retention signals (SEQ ID NO: 10).

FIGS. 7-11 represent one destination vector, pDAB3736 ( FIG. 7 ), and four donor vectors, pDAB3912 ( FIG. 8 ), pDAB3914 ( FIG. 9 ), pDAB3916 ( FIG. 10 ), and pDAB3724 ( FIG. 11 ) used to build nine binary constructs with the Gateway™ technology.

FIG. 12 depicts Gateway™ WNV ME binary vector, pDAB3920. pDAB3920 encodes T-DNA Border B/RB7 MAR v3/CsVMV promoter v2/WNV ME v2/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 13 illustrates Gateway™ binary vector, pDAB3922. pDAB3922 contains the following elements: T-DNA Border B/RB7 MAR v3/AtuMAS 4OCS promoter v4/15 kDa zein ER v2-WNV ME v2-KDELv3/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 14 represents Gateway™ West Nile Virus binary vector, pDAB3924. The pDAB3924 vector contains the following elements: T-DNA Border B/RB7 MAR v3/At Ubi10 promoter (Genbank Accession no L05363) v2/15 kDa zein ER v2-WNV ME v2-KDEL v3/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 15 pertains to a Gateway™ binary vector, pDAB3927 containing the following elements: T-DNA Border B/RB7 MAR v3/CsVMV promoter v2/15 kDa zein ER signal v2-WNV ME v2/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 16 provides Gateway™ binary vector, pDAB3929. pDAB3929 contains T-DNA Border B/RB7 MAR v3/CsVMV promoter v2/Nt osm 5′ UTR v3/15 kDa zein ER v2-WNV ME v2-KDEL v3/Nt osm 3′ UTR v3/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 17 is Gateway™ binary vector, pDAB3934. This vector contains the following elements: T-DNA Border B/RB7 MAR v3/ORF25/26 3′ UTR/KDELv3/WNV ME v3/15 kDa zein ER signal v2 (SEQ ID NO: 14)/AtuMAS 4OCS promoter v4/15 kD zein ER signal v2-WNV ME v2-KDELv3/Atu ORF23 3, UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 18 provides a depiction of Gateway™ binary vector, pDAB3941. pDAB3941 contains the following components: T-DNA Border B/RB7 MAR v3/CsVMV promoter v2/15 kD zein ER v2-WNV ME v2-KDEL v3/Atu ORF23 3′UTR v1/AtUbi3 promoter v2/15 kD zein ER v2-WNV ME v3-KDELv3/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 19 provides Gateway™ binary vector, pDAB3943. This vector contains the following elements: T-DNA Border B/RB7 MAR v3/CsVMVv2/WNV M v2 E with modified glycosylation site (v5)/Atu ORF23 3′ UTR v1/AtUbi10 promoter v2/PAT v3/Atu ORF1 3′ UTR v3/Multiple T-DNA Border A.

FIG. 20 provides E protein expression of 14 Day callus events transformed with pDAB2475 (ER targeted, ME Version 2, KDEL), as detected by ELISA.

FIG. 21 provides E protein expression of 14 Day callus events transformed with pDAB2478 (ER targeted, prME Version 2, KDEL) as detected by ELISA.

FIG. 22 provides E protein expression of 14 Day callus events transformed with pDAB2481 (ER targeted, prME with modified glycosylation site (Version 4), KDEL) as detected by ELISA.

FIG. 23 compares the expression levels between events transformed with pDAB2475, pDAB2478, and pDAB2481. A significantly higher protein recovery potential from pDAB2475 is indicated in the figure.

FIG. 24 depicts samples from select events that were analyzed by Western blot (day 14 callus). From many of the pDAB2475 events, full-length E protein was detected at the expected ˜54 kDa size of the authentic mature virion E protein.

FIGS. 25 and 26 illustrate that fewer events expressing the full-length E protein were detected with the pDAB2478 and pDAB2481 constructs.

FIG. 27 compares ELISA Results from Day 14 Callus of All Events of pDAB3920, pDAB3922, pDAB3924, pDAB3927, pDAB3929, pDAB3943, pDAB3934 and pDAB3941.

FIG. 28 depicts 14 Day callus samples from events of pDAB3920 and pDAB3922 analyzed by Western blot.

FIG. 29 depicts 14 Day callus samples from events of pDAB3924 and pDAB3927 analyzed by Western blot.

FIG. 30 depicts 14 Day callus samples from events of pDAB3929 and pDAB3934 analyzed by Western blot.

FIG. 31 illustrates on-line fermentation profiles for WNV event 1622-207 during a 10 liter STR fermentation run (Batch ID WNV SRD05006). The reduction in agitator speed rate resulted in the decrease in oxygen uptake rate near the termination of the fermentation.

FIG. 32 provides a fermentation residuals analysis for batch ID WNV SRD05006.

FIG. 33 provides a fermentation residuals analysis for batch ID WNV SRD05007.

FIG. 34 illustrates the kinetics of ME production in N. tobacum NT-1 suspension cells as determined over a period of 9 days for recombinant West Nile Virus events 1622-207 and 1622-210. Production of WNV envelope protein during a 218 hour (9.08 day; subtract the 42 hour pre-inoculation phase from the x-axis time) 10 liter stirred-tank reactor fermentation is depicted. The maximum volumetric productivity of ME events 1622-210 and 1622-207 occurred at 164 hr (206-42 hr), and 188 hr (230-42 hr) post-inoculation respectively.

FIG. 35 provides a graphical presentation of WNV serum neutralizing titers from a mouse clinical model study (Study I). The figure was generated by changing neutralization titers of >2560 to 2560 and titers of <20 to 20 and calculating serum neutralization geometric mean titer (GMT) for each treatment group.

FIG. 36 shows the variable response demonstrated by different doses of antigen and formulation with different adjuvants (Study II).

SEQ ID NO: 1 is a native DNA sequence of flamingo isolate of West Nile Virus from GenBank Accession AF196835, encoding prM-, M-, and E-peptides (Version 1). The native WNV prM-M-E peptide coding region is 2004 bases in length and encodes the prM peptide (bases 1-276), the M-peptide (bases 277-501) and the E-Peptide (bases 502-2004).

SEQ ID NO: 2 is an amino acid sequence of native prM-, M-, and E-peptides encoded by SEQ ID NO: 1. The prM peptide is amino acids 1-92, the M-peptide is amino acids 93-167 and the E-peptide is amino acids 168-668.

SEQ ID NO: 3 is a tobacco-optimized DNA sequence for prM-, M- and E-peptides (Version 2). SEQ ID NO: 3 is 2004 bases in length and the prM-peptide is encoded by bases 1-276, the M-peptide is encoded by bases 277-501 and the E-Peptide encoded by bases 502-2004.

SEQ ID NO: 4 is a tobacco-optimized DNA sequence for prM-, M- and E-peptides with mutated N-glycosylation site (Version 4). The proline codon is at nts 967-969 and the sequence is 2004 bases in length. The prM-peptide is encoded by bases 1-276, the M-peptide encoded by bases 277-501 and the E-Peptide encoded by bases 502-2004.

SEQ ID NO: 5 is an amino acid sequence of prM-, M-, and E-peptides encoded by SEQ ID NO: 4 and containing a mutated N-glycosylation site. The proline residue is at position 323 and the sequence is 668 amino acids in length. The prM-peptide is amino acids 1-92, the M-peptide is amino acids 93-167 and the E-peptide is amino acids 168-668.

SEQ ID NO: 6 is a tobacco-optimized DNA sequence encoding M- and E-peptides (Version 2). The sequence is 1728 bases in length and the M-peptide is encoded by bases 1-225. The E-Peptide is encoded by bases 226-1728.

SEQ ID NO: 7 is a tobacco-optimized DNA sequence encoding M- and E-peptides (Version 3). This sequence is 1728 bases in length and the M-peptide is encoded by bases 1-225. The E-peptide is encoded by bases 226-1728.

SEQ ID NO: 8 is a tobacco-optimized DNA sequence encoding chimeric protein including 15 kDa zein ER targeting signal peptide, prM-, M- and E-peptides (Version 2), and KDEL. The sequence is 2106 bases in length and the 15 kDa ER targeting signal is encoded by bases 1-66. The prM-peptide is encoded by bases 67-342, the M-peptide is encoded by bases 343-567, the E-peptide is encoded by bases 568-2070, the KDEL ER retention signal is encoded by bases 2071-2082 and six frame stops are located at bases 2083-2106.

SEQ ID NO: 9 is an amino acid sequence of the chimeric fusion protein encoded by SEQ ID NO: 8. The fusion protein is 694 amino acids in length and contains a 15 kDa zein ER targeting peptide (amino acids 1-22), the prM-peptide (amino acids 23-114), the M-peptide (amino acids 115-189), the E-peptide (amino acids 190-690), an N-glycosylation site (amino acids 343-345) and the KDEL ER retention signal (amino acids 691-694).

SEQ ID NO: 10 is a tobacco-optimized DNA sequence encoding chimeric protein including 15 kDa zein ER targeting signal peptide, prM-, M- and E-peptides with mutated N-glycosylation site (Version 4) and KDEL. The sequence is 2106 bases in length and the kDa ER targeting signal is encoded by bases 1-66, the prM-peptide is encoded by bases 67-342, the M-peptide is encoded by bases 343-567, the E-peptide is encoded by bases 568-2070, the KDEL ER retention signal is encoded by bases 2071-2082 and six frame stops are located at bases 2083-2106.

SEQ ID NO: 11 is an amino acid sequence of the chimeric fusion protein encoded by SEQ ID NO: 10. The polypeptide is 694 amino acids in length and the 15 kDa zein ER targeting peptide is located at amino acids 1-22. The prM-peptide is found at amino acids 23-114, the M-peptide is found at amino acids 115-189, the E-peptide is found at amino acids 190-690 and mutated N-glycosylation site is at amino acids 343-345 and the KDEL ER retention signal is amino acids 691-694.

SEQ ID NO: 12 is a tobacco-optimized DNA sequence encoding chimeric protein including 15 kDa zein ER targeting signal peptide, M- and E-peptides (Version 2) and KDEL. The sequence is 1830 bases in length and the 15 kDa ER targeting signal is encoded by bases 1-66, the M-peptide is encoded by bases 67-291, the E-peptide is encoded by bases 292-1794, the KDEL ER retention signal is encoded by bases 1795-1806 and the six frame stops comprise bases 1807-1830.

SEQ ID NO: 13 is an amino acid sequence of the chimeric fusion protein encoded by SEQ ID NO: 12. This sequence is 602 amino acids long and the 15 kDa zein ER targeting peptide is amino acids 1-22. The M-peptide is located at amino acids 23-97, the E-peptide is located at amino acids 98-598 and the KDEL ER retention signal is found at amino acids 599-602.

SEQ ID NO: 14 is a tobacco-optimized DNA sequence encoding chimeric protein including 15 kDa zein ER targeting signal peptide, M- and E-peptides (Version 3) and KDEL. This sequence is 1832 bases in length, the 15 kDa ER targeting signal is encoded by bases 6-68, the M-peptide is encoded by bases 69-293, the E-peptide is encoded by bases 294-1796, the KDEL ER retention signal is encoded by bases 1797-1808 and six frame stops comprise bases 1809-1832.

SEQ ID NO: 15 is an amino acid sequence of the chimeric fusion protein encoded by SEQ ID NO: 14. The sequence is 601 amino acids in length and the 15 kDa zein ER targeting peptide is amino acids 1-21. The M-peptide is located at amino acids 22-96, the E-peptide is located at amino acids 97-597 and the KDEL ER retention signal is found at amino acids 598-601.

The subject application provides the following non-limiting compositions of matter as well as methods of using these compositions of matter in the production of immunogenic polypeptides and methods of inducing immune responses in individuals. Thus, the subject invention provides various compositions of matter comprising:

a) isolated, purified, and/or recombinant polypeptides comprising SEQ ID NO: 5, 9, 11, 13 or 15;

b) a fragment of the polypeptide set forth in SEQ ID NO: 5, 9, 11, 13, 15 or a fragment of SEQ ID NO: 5, 9, 11, 13 or 15 that is “from Y to Z”, wherein Y is the N-terminal amino acid of the specified sequence and Z is the C-terminal amino acid of the specified sequence. Thus, for SEQ ID NO: 5, each fragment can be between 5 consecutive amino acids and 667 consecutive amino acids in length. Each fragment containing between 5 and 693 consecutive amino acids of SEQ ID NO: 9 and 11 are specifically contemplated by the subject invention. Likewise, for SEQ ID NO: 13, each polypeptide fragment between 5 and 601 consecutive amino acids is specifically contemplated by the subject invention. Further, each polypeptide fragment spanning between 5 and 600 consecutive amino acids of SEQ ID NO: 15 is also specifically contemplated by the subject invention. Fragments “from Y to Z”, wherein Y is the N-terminal amino acid of the specified sequence and Z is the C-terminal amino acid of a specified sequence are provided in Table 9 for SEQ ID NO: 5, Table 10 for SEQ ID NOs: 9 and 11, Table 11 for SEQ ID NO: 13 and Table 12 for SEQ ID NO: 15. Polypeptide fragments as set forth in this application have at least one biological activity that is substantially the same as the corresponding biological activity of the full-length polypeptide of SEQ ID NO: 5, 9, 11, 13 or 15 Various other exemplary polypeptide fragments are set forth in Tables 15 or 16;

c) an E-peptide as set forth in any one of SEQ ID NOs: 5, 9, 11, 13 or 15 or a fragment of an E-peptide as set forth in any one of SEQ ID NOs: 5, 9, 11, 13 or 15 that produces a neutralizing antibody response when administered to an individual;

d) a polypeptide according to any one of embodiments a), b) or c) that further comprises a heterologous polypeptide sequence;

e) a plant-derived polypeptide according to any one of embodiments a), b), c) or d);

f) a composition comprising a carrier and a polypeptide as set forth in any one of a), b), c), d) or e), wherein said carrier is cellular material from the plant, mammalian or bacterial expression system (optionally suspended in a buffer), an adjuvant or a pharmaceutically acceptable excipient;

g) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15 or encoding one or more polypeptide fragment of SEQ ID NOs: 5, 9, 11, 13 or 15 as set forth in (b) or (c), optionally wherein said polynucleotide sequence has a G+C content of at least 40% and less than 50% or a G+C content as set forth in Table 13;

h) a polynucleotide sequence that is at least 70% (or a percentage as specified in the Table 14) identical to SEQ ID NO: 1, encodes a polypeptide comprising SEQ ID NO: 2 and has a G+C content of between about 40% and about 50% (or a specific G+C content as specified in Table 13);

i) a polynucleotide sequence at least 8 consecutive nucleotides of a polynucleotide sequence as set forth in (g) or (h);

j) a polynucleotide sequence comprising SEQ ID NO: 3, 4, 6, 7, 8, 10, 12, or 14 or a fragment of at least 8 consecutive nucleotides of SEQ ID NO: 3, 4, 6, 7, 8, 10, 12, or 14;

k) a polynucleotide that is complementary to the polynucleotides set forth in (g), (h), (i), or (j);

l) a polynucleotide that hybridizes under low, intermediate or high stringency with a polynucleotide sequence as set forth in (g), (h), (i), (i) or (k);

m) a genetic construct comprising a polynucleotide sequence as set forth in (g), (h), (i), (i) or (k);

n) a vector comprising a polynucleotide or genetic construct as set forth in (g), (h), (i), (i), (j), (k) or (l);

o) a host cell comprising a vector as set forth in (n), a genetic construct as set forth in (m), or a polynucleotide as set forth in any one of (g), (h), (i), (j) or (k);

p) a transgenic plant, plant cell, or plant part comprising a vector as set forth in (n), a genetic construct as set forth in (m) or a polynucleotide as set forth in any one of (g), (h), (i), (j) or (k); or

q) a probe comprising a polynucleotide according to (g), (h), (i), (j), (k) or (l) and, optionally, a label or marker.

In the context of the instant invention, the terms “oligopeptide”, “polypeptide”, “peptide” and “protein” can be used interchangeably; however, it should be understood that the invention does not relate to the polypeptides in natural form, that is to say that they are not in their natural environment but that the polypeptides may have been isolated or obtained by purification from natural sources or obtained from host cells prepared by genetic manipulation (e.g., the polypeptides, or fragments thereof, are recombinantly produced by host cells, or by chemical synthesis). Polypeptides according to the instant invention may also contain non-natural amino acids, as will be described below. The terms “oligopeptide”, “polypeptide”, “peptide” and “protein” are also used, in the instant specification, to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Linker elements can be joined to the polypeptides of the subject invention through peptide bonds or via chemical bonds (e.g., heterobifunctional chemical linker elements) as set forth below. Additionally, the terms “amino acid(s)” and “residue(s)” can be used interchangeably.

In the context of both polypeptides and polynucleotides, the term “successive” can be used interchangeably with the term “consecutive” or the phrase “contiguous span” throughout the subject application. Thus, in some embodiments, a polynucleotide fragment may be referred to as “a contiguous span of at least X nucleotides, wherein X is any integer value beginning with 5; the upper limit for fragments as set forth herein is one nucleotide less than the total number of nucleotides found in the full-length sequence encoding a particular polypeptide (e.g., a polypeptide comprising SEQ ID NO: 9). A polypeptide fragment, by example, may be referred to as “a contiguous span of at least X amino acids, wherein X is any integer value beginning with 5; the upper limit for such polypeptide fragments is one amino acid less than the total number of amino acids found in the full-length sequence of a particular polypeptide (e.g., 667 for SEQ ID NO: 5, 693 for SEQ ID NO: 9 and 11, 601 amino acids for SEQ ID NO: 13 and 600 amino acids for SEQ ID NO: 15). As used herein, the term “integer” refers to whole numbers in the mathematical sense.

“Nucleotide sequence”, “polynucleotide” or “nucleic acid” can be used interchangeably and are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g., RNA molecules). It should also be understood that the present invention does not relate to genomic polynucleotide sequences in their natural environment or natural state. The nucleic acid, polynucleotide, or nucleotide sequences of the invention can be isolated, purified (or partially purified), by separation methods including, but not limited to, ion-exchange chromatography, molecular size exclusion chromatography, or by genetic engineering methods such as amplification, subtractive hybridization, cloning, subcloning or chemical synthesis, or combinations of these genetic engineering methods. The terms “polynucleotide vaccine” and “DNA vaccine” can also be used interchangeably herein.

The terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term. The phrases “isolated” or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. “Link” or “join” refers to any method known in the art for functionally connecting peptides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.

Thus, the subject invention provides polypeptides comprising SEQ ID NOs: 5, 9, 11, 13 or 15 and/or polypeptide fragments of SEQ ID NOs: 5, 9, 11, 13 or 15. Polypeptide fragments, according to the subject invention, comprise a contiguous span of at least 5 consecutive amino acids of SEQ ID NOs: 5, 9, 11, 13 or 15. Polypeptide fragments according to the subject invention can be any integer in length from at least 5 consecutive amino acids to 1 amino acid less than a full length polypeptide of SEQ ID NO: 5, 9, 11, 13 or 15. Fragments of SEQ ID NO: 5 can contain any number (integer) of consecutive amino acids between, and including, 5 and 667. For SEQ ID NO: 9 or 11 a polypeptide fragment is any number (integer) of consecutive amino acids between, and including, 5 and 693. For SEQ ID NO: 13, a polypeptide fragment is any number (integer) of consecutive amino acids between, and including, 5 and 601. For SEQ ID NO: 15, a polypeptide fragment is any number (integer) of consecutive amino acids between, and including 5 and 600 amino acids.

Each polypeptide fragment of the subject invention can also be described in terms of its N-terminal and C-terminal positions. Additionally, polypeptide fragments embodiments described herein may be “at least”, “equal to”, “equal to or less than”, “less than”, “at least _ but not greater than _” or “from Y to Z”, wherein Y is the N-terminal amino acid of the specified sequence and Z is the C-terminal amino acid of the specified sequence, the fragment is at least 5 amino acids in length, and Y and Z are any integer specified (or selected from) those integers identified in the tables specifying the corresponding fragment lengths for each polypeptide disclosed herein (see Tables 9, 10, 11, 12, 15, and 16 [the positions listed in the tables correspond to the amino acid position as provided in the attached sequence listing]). As is apparent from Table 10, the N-terminal amino acid for fragments of SEQ ID NOs: 9 and 11 can be any integer from 1 to 690 and the C-terminal amino acid is any integer from 5 to 694 (depending on the fragment length which is to be any number (integer) of consecutive amino acids between, and including, 5 and 694). For fragments of SEQ ID NO: 5 (shown in Table 9), the N-terminal amino acid can be any integer between 1 and 664 and the C-terminal amino acid is any integer from 5 to 667 (depending on the fragment length which is to be any number (integer) of consecutive amino acids between, and including, 5 and 667). With respect to fragments of SEQ ID NO: 13 (illustrated in Table 11), the N-terminal amino acid can be any integer between 1 and 598 and the C-terminal amino acid is any integer from 5 to 602 (depending on the fragment length which is any number (integer) of consecutive amino acids between, and including, 5 and 601 amino acids). For SEQ ID NO: 15 (provided in Table 12), the N-terminal amino acid can be any integer between 1 and 597 and the C-terminal amino acid is any integer from 5 to 601 (depending on the fragment length which is any number (integer) of consecutive amino acids between, and including, 5 and 600 amino acids). It is noted that all ranges used to describe any embodiment of the present invention are inclusive unless specifically set forth otherwise and that fragments of a given polypeptide can be any integer in length, provided that the length of the polypeptide fragment is at least one amino acid shorter than the polypeptide identified in SEQ ID NO: 5, 9, 11, 13 or 15. To illustrate this concept, the four fragments provided by Table 12 that are 598 amino acids in length are provided. Thus, the various polypeptide fragments are defined as: where Y is position 1 of SEQ ID NO: 15, Z is position 598 of SEQ ID NO: 15 (the peptide is 598 amino acids in length); where Y is position 2 of SEQ ID NO: 15, Z is position 599 of SEQ ID NO: 15 (the peptide is 598 amino acids in length); where Y is position 3 of SEQ ID NO: 15, Z is position 600 of SEQ ID NO: 15 (the peptide is 598 amino acids in length); and where Y is position 4 of SEQ ID NO: 15, Z is position 601 of SEQ ID NO: 15 (the peptide is 598 amino acids in length).

The subject invention also provides for various polypeptide fragments (comprising contiguous spans or consecutive spans of at least five consecutive amino acids) that span particular residues of SEQ ID NO: 5, 9, 11, 13 or 15. For SEQ ID NOs: 9 and 11, preferred fragments include those of at least five consecutive amino acids that include at least one of the amino acids at positions 1-22 [i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or all 22 of the amino acids], at least one, two or all three of the amino acids at positions 343-345 of SEQ ID NOs: 9 or 11, and at least one, two, three or all four of amino acids 691 through 694 as set forth in SEQ ID NO: 9 or 11. Non-limiting examples illustrating a few of these combinations of amino acids are set forth in Tables 15 or 16. For SEQ ID NO: 5, certain embodiments provide for any of those fragments of at least five consecutive amino acids that span amino acid 323. For SEQ ID NO: 13, various embodiments of the invention provide polypeptide fragments of at least five consecutive amino acids that span or include: at least one of the amino acids at positions 1-22 of SEQ ID NO: 13; and/or at least one, two, three, or all four of the amino acids at positions 599-602 of SEQ ID NO: 13. With respect to SEQ ID NO: 15, exemplary polypeptide fragments include those that span, or include at least one of the amino acids at positions 1-21 and/or 598-601 of SEQ ID NO: 15. Additional polypeptide fragments are also set forth in Tables 15 and 16. In some aspects of the invention, preferred polypeptide fragments are the complete E-peptide sequence identified in SEQ ID NOs: 5, 9, 11, 13 or 15.

Fragments, as described herein, can be obtained by cleaving the polypeptides of the invention with a proteolytic enzyme (such as trypsin, chymotrypsin, or collagenase) or with a chemical reagent, such as cyanogen bromide (CNBr). Alternatively, polypeptide fragments can be generated in a highly acidic environment, for example at pH 2.5. Such polypeptide fragments may be equally well prepared by chemical synthesis or using hosts transformed with an expression vector according to the invention. The transformed host cells contain a nucleic acid, allowing the expression of these fragments, under the control of appropriate elements for regulation and/or expression of the polypeptide fragments.

In certain preferred embodiments, fragments of the polypeptides disclosed herein retain at least one biological property or biological activity of the full-length polypeptide from which the fragments are derived (such fragments may also be referred to as “biologically active fragments”. Thus, both full length polypeptides and fragments of the polypeptides provided by SEQ ID NO: 5, 9, 11, 13 or 15 have one or more of the following properties or biological activities: the ability to: 1) specifically bind to antibodies specific for SEQ ID NO: 5, 9, 11, 13 or 15; 2) specifically bind antibodies found in an animal or human infected with West Nile virus and/or antibodies that neutralize West Nile infectious virus (the ability of the virus to infect a host or target cell); the ability to bind to, and activate T-cell receptors (CTL (cytotoxic T-lymphocyte) and/or HTL (helper T-lymphocyte receptors)) in the context of MHC Class I or Class II antigen that are isolated or derived from an animal or human infected with West Nile virus; 3) the ability to induce an immune response in an animal or human against a West Nile virus; 4) the ability to induce a protective immune response in an animal or human against a West Nile virus; and/or 5) the ability to induce the production of West Nile Virus neutralizing antibodies (also referred to a neutralizing antibodies) in an animal/individual immunized with one or more of said polypeptides.

Where plant expression systems are used for the production of polypeptides provided in the subject application, or fragments thereof, a composition comprising the purified polypeptide can include plant cell components (e.g., cell walls, the cellular matrix of plant cell membranes and carbohydrates, etc.) or plant cell matrix components. Likewise, where eukaryotic or prokaryotic expression systems are used for the production of polypeptides of the subject invention, or fragments thereof, cell membrane or cell wall components of each respective expression system may be present in a composition comprising partially purified polypeptides.

The polypeptides (or fragments thereof) of the invention may be monomeric or multimeric (e.g., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions containing them. Multimeric polypeptides, as set forth herein, may be formed by hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. One non-limiting example of such a covalent association is the formation of disulfide bonds between immunoglobulin heavy chains as provided by a fusion protein of the invention that comprises a polypeptide comprising SEQ TD NO: 5, 9, 11, 13 or 15 (or fragments thereof) fused to an Ig heavy chain (see, e.g., U.S. Pat. No. 5,478,925, which disclosure is hereby incorporated by reference in its entirety). Another example of a fusion protein capable of forming covalently associated multimers is oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Other multimeric polypeptides can be formed by fusing the polypeptides of the invention to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Non-limiting examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

Multimeric polypeptides can also be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimeric polypeptides can be generated by introducing disulfide bonds between the cysteine residues located within the sequence of the polypeptides that are being used to construct the multimeric polypeptide (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, other techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

The polypeptides provided herein, as well as the fragments thereof, may further comprise linker elements (L) that facilitate the attachment of the fragments to other molecules, amino acids, or polypeptide sequences. The linkers can also be used to attach the polypeptides, or fragments thereof, to solid support matrices for use in affinity purification protocols. Non-limiting examples of “linkers” suitable for the practice of the invention include chemical linkers (such as those sold by Pierce, Rockford, Ill.), or peptides that allow for the connection combinations of polypeptides (see, for example, linkers such as those disclosed in U.S. Pat. Nos. 6,121,424, 5,843,464, 5,750,352, and 5,990,275, hereby incorporated by reference in their entirety).

In other embodiments, the linker element (L) can be an amino acid sequence (a peptide linker). In some embodiments, the peptide linker has one or more of the following characteristics: a) it allows for the free rotation of the polypeptides that it links (relative to each other); b) it is resistant or susceptible to digestion (cleavage) by proteases; and c) it does not interact with the polypeptides it joins together. In various embodiments, a multimeric construct according to the subject invention includes a peptide linker and the peptide linker is 5 to 60 amino acids in length. More preferably, the peptide linker is 10 to 30, amino acids in length; even more preferably, the peptide linker is 10 to 20 amino acids in length. In some embodiments, the peptide linker is 17 amino acids in length.

Peptide linkers suitable for use in the subject invention are made up of amino acids selected from the group consisting of Gly, Ser, Asn, Thr and Ala. Preferably, the peptide linker includes a Gly-Ser element. In a preferred embodiment, the peptide linker comprises (Ser-Gly-Gly-Gly-Gly) y wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. Other embodiments provide for a peptide linker comprising ((Ser-Gly-Gly-Gly-Gly) y -Ser-Pro). In certain preferred embodiments, y is a value of 3, 4, or 5. In other preferred embodiment, the peptide linker comprises (Ser-Ser-Ser-Ser-Gly) y or ((Ser-Ser-Ser-Ser-Gly) y -Ser-Pro), wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. In certain preferred embodiments, y is a value of 3, 4, or 5. Where cleavable linker elements are desired, one or more cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) can be used alone or in combination with the aforementioned linkers.

Multimeric constructs of the subject invention can also comprise a series of repeating elements, optionally interspersed with other elements. As would be appreciated by one skilled in the art, the order in which the repeating elements occur in the multimeric polypeptide is not critical and any arrangement of the repeating elements as set forth herein can be provided by the subject invention. Thus, a “multimeric construct” according to the subject invention can provide a multimeric polypeptide comprising a series of polypeptides or polypeptide fragments that are, optionally, joined together by linker elements (either chemical linker elements or amino acid linker elements).

Fusion proteins according to the subject invention comprise one or more heterologous polypeptide sequences (e.g., tags that facilitate purification of the polypeptides of the invention (see, for example, U.S. Pat. No. 6,342,362, hereby incorporated by reference in its entirety; Altendorf et al., (1999-WWW, 2000); Baneyx, (1999); Eihauer et al., (2001); Jones et al. (1995); Margolin (2000); Puig et al., (2001); Sassenfeld (1990); Sheibani (1999); Skerra et al., (1999); Smith (1998); Smyth et al., (2000); Unger (1997), each of which is hereby incorporated by reference in their entireties), or commercially available tags from vendors such as STRATAGENE (La Jolla, Calif.), NOVAGEN (Madison, Wis.), QIAGEN, Inc., (Valencia, Calif.), or InVitrogen (San Diego, Calif.).

In other embodiments, polypeptides of the subject invention (e.g., SEQ ID NOs: 5, 9, 11, 13, 15 or fragments thereof) can be fused to heterologous polypeptide sequences that have adjuvant activity (a polypeptide adjuvant). Non-limiting examples of such polypeptides include heat shock proteins (hsp) (see, for example, U.S. Pat. No. 6,524,825, the disclosure of which is hereby incorporated by reference in its entirety).

The subject invention also provides biologically active fragments of a polypeptide according to the invention and includes those peptides capable of eliciting an immune response directed against a West Nile virus, said immune response providing components (B-cells, antibodies, and/or components of the cellular immune response (e.g., helper, cytotoxic, and/or suppressor T-cells)) reactive with the fragment of said polypeptide; the intact, full length, unmodified polypeptide disclosed herein; or both a fragment of a polypeptide and the intact, full length, unmodified polypeptides disclosed herein. Certain embodiments provide methods of inducing an antibody response that produces West Nile virus neutralizing antibodies.

The subject application also provides a composition comprising at least one isolated, recombinant, or purified polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15 (or a fragment thereof) and at least one additional component. In various aspects of the invention, the additional component is a solid support (for example, microtiter wells, magnetic beads, non-magnetic beads, agarose beads, glass, cellulose, plastics, polyethylene, polypropylene, polyester, nitrocellulose, nylon, or polysulfone). The additional component can also be a pharmaceutically acceptable excipient or adjuvant known to those skilled in the art. In some aspects of the invention, the solid support provides an array of polypeptides of the subject invention or an array of polypeptides comprising combinations of various polypeptides of the subject invention. Other aspects of the invention provide a composition comprising the purified polypeptide that includes plant cell components (e.g., cell walls, the cellular matrix of plant cell membranes and carbohydrates, etc.) or plant cell matrix components. Likewise, where eukaryotic or prokaryotic expression systems are used for the production of polypeptides or fragments of the polypeptides provided by this application, cell membrane or cell wall components of each respective expression system may be present in a composition comprising partially purified polypeptides.

The subject invention also provides methods for eliciting an immune response in an individual comprising the administration of compositions comprising polypeptides according to the subject invention to an individual in amounts sufficient to induce an immune response in the individual. In some embodiments, a “protective” or “therapeutic immune response” is induced in the individual. A “protective immune response” or “therapeutic immune response” refers to an induction in the production of antibodies that neutralize infectious West Nile viruses, or induce a CTL (or CD8 + T cell) and/or an HTL (or CD4 + T cell), and/or an antibody response that prevents, reduces or at least partially arrests disease symptoms, side effects or progression in the individuals. For example, individuals in which a protective immune response has been induced can exhibit reduced mortality and/or exhibit reduced viral shedding as compared to non-immunized control individuals. The protective immune response may also include an antibody response that has been facilitated by the stimulation of helper T cells (or CD4 + T cells). Additional methods of inducing an immune response in an individual are taught in U.S. Pat. No. 6,419,931, hereby incorporated by reference in its entirety. The term CTL can be used interchangeably with CD8 + T-cell(s) and the term HTL can be used interchangeably with CD4 + T-cell(s) throughout the subject application.

Individuals, in the context of this application, refers to birds and/or mammals such as, but not limited to, apes, chimpanzees, orangutans, humans, monkeys or domesticated animals (pets) such as dogs, cats, guinea pigs, hamsters, rabbits, ferrets, cows, horses, goats and sheep. Avian or bird is herein defined as any warm-blooded vertebrate member of the class Aves typically having forelimbs modified into wings, scaly legs, a beak, and bearing young in hard-shelled eggs. For purposes of this specification, preferred groups of birds are domesticated chickens, turkeys, ostriches, ducks, geese, swan, Cornish game hens and exotic birds kept as pets or for display in zoos.

Administering or administer is defined as the introduction of a substance into the body of an individual and includes oral, nasal, ocular, rectal, vaginal and parenteral routes. Compositions may be administered individually or in combination with other agents via any route of administration, including but not limited to subcutaneous (SQ), intramuscular (IM), intravenous (IV), intraperitoneal (IP), intradermal (ID), transdermal, (TD), or via the nasal, ocular, oral, or rectal mucosa.

The composition administered to the individual may, optionally, contain an adjuvant and may be delivered in any manner known in the art for the delivery of immunogen to a subject. Compositions may also be formulated in any carriers, including for example, pharmaceutically acceptable carriers such as those described in E. W. Martin's Remington's Pharmaceutical Science , Mack Publishing Company, Easton, Pa. In preferred embodiments, compositions may be formulated in incomplete Freund's adjuvant, complete Freund's adjuvant, or alum. Other non-limiting examples of adjuvants that can be used in the practice of the invention include: oil-water emulsions, Polygen, Carbigen (Carbopol 974P NF) or Titer-Max (Block copolymer CRL-8941, squalene and a unique microparticulate stabilizer).

In other embodiments, the subject invention provides for diagnostic assays based upon Western blot formats or standard immunoassays known to the skilled artisan and which utilize a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 5, 9, 11, 13 or 15. For example, antibody-based assays such as enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), lateral flow assays, reversible flow chromatographic binding assay (see, for example, U.S. Pat. No. 5,726,010, which is hereby incorporated by reference in its entirety), immunochromatographic strip assays, automated flow assays, and assays utilizing peptide-containing biosensors may be employed for the detection of antibodies that bind to the polypeptides (or fragments thereof) that are provided by the subject invention. The assays and methods for conducting the assays are well-known in the art and the methods may test biological samples (e.g., serum, plasma, or blood) qualitatively (presence or absence of antibody) or quantitatively (comparison of a sample against a standard curve prepared using a polypeptide of the subject invention) for the presence of antibodies that bind to polypeptides of the subject invention.

The antibody-based assays can be considered to be of four types: direct binding assays, sandwich assays, competition assays, and displacement assays. In a direct binding assay, either the antibody or antigen is labeled, and there is a means of measuring the number of complexes formed. In a sandwich assay, the formation of a complex of at least three components (e.g., antibody-antigen-antibody) is measured. In a competition assay, labeled antigen and unlabelled antigen compete for binding to the antibody, and either the bound or the free component is measured. In a displacement assay, the labeled antigen is pre-bound to the antibody, and a change in signal is measured as the unlabelled antigen displaces the bound, labeled antigen from the receptor.

Lateral flow assays can be conducted according to the teachings of U.S. Pat. No. 5,712,170 and the references cited therein. U.S. Pat. No. 5,712,170 and the references cited therein are hereby incorporated by reference in their entireties. Displacement assays and flow immunosensors useful for carrying out displacement assays are described in: Kusterbeck et al., (1990); Kusterbeck et al., (1990a); Ligler et al., (1992); Ogert et al., (1992), all of which are incorporated herein by reference in their entireties. Displacement assays and flow immunosensors are also described in U.S. Pat. No. 5,183,740, which is also incorporated herein by reference in its entirety. The displacement immunoassay, unlike most of the competitive immunoassays used to detect small molecules, can generate a positive signal with increasing antigen concentration.

The subject invention also provides methods of binding an antibody to a polypeptide of the subject invention (e.g., SEQ ID NO: 5, 9, 11, 13 or 15, or an antibody binding fragment thereof) comprising contacting a sample containing an antibody with a polypeptide under conditions that allow for the formation of an antibody-antigen complex. These methods can further comprise the step of detecting the formation of said antibody-antigen complex. In various aspects of this method, an immunoassay is conducted for the detection of West Nile virus specific antibodies in a sample. Non-limiting examples of such immunoassays include enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), lateral flow assays, immunochromatographic strip assays, automated flow assays, Western blots, immunoprecipitation assays, reversible flow chromatographic binding assays, agglutination assays, and biosensors. Additional aspects of the invention provide for the use of an array of polypeptides when conducting the aforementioned methods of detecting antibodies specific to West Nile virus (the array can contain at least one of the polypeptides set forth in SEQ ID NOs: 5, 9, 11, 13 or 15 (or fragments thereof) and can also contain other polypeptides of the same or different viral origin).

The subject invention also concerns antibodies that bind to polypeptides of the invention. Antibodies that are immunospecific for the polypeptides as set forth herein are specifically contemplated. In various embodiments, antibodies that do not cross-react with other known West Nile virus polypeptides are preferred. Particularly preferred antibodies do not cross-react with antibodies produced against polypeptides derived from known strains of West Nile virus. The antibodies of the subject invention can be prepared using standard materials and methods known in the art (see, for example, Monoclonal Antibodies. Principles and Practice, 1983 ; Monoclonal Hybridoma Antibodies: Techniques and Applications, 1982 ; Selected Methods in Cellular Immunology, 1980 ; Immunological Methods, Vol. II, 1981 ; Practical Immunology , and Kohler et al., 1975; Letchworth and Appleton, 1984). These antibodies can further comprise one or more additional components, such as a solid support, a carrier or pharmaceutically acceptable excipient, or a label.

The term “antibody” includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity, particularly neutralizing activity. “Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments. Particularly preferred antibodies according to the subject invention are those that do not bind to the unmodified WNV polypeptides known in the art.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) and Marks et al. (1991), for example.

The monoclonal antibodies described herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., (1984)). Also included are humanized antibodies that specifically bind to the polypeptides, or fragments thereof, set forth in SEQ ID NO: 5, 9, 11, 13 or 15 (see, for example, U.S. Pat. No. 6,407,213 or 6,417,337, which are hereby incorporated by reference in their entirety, teaching methods of making humanized antibodies).

“Single-chain Fv” or “sFv” antibody fragments comprise the V H and V T domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun (1994).

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L ). Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Holliger et al. (1993). The term “linear antibodies” refers to the antibodies described in Zapata et al. (1995).

An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

As discussed above, “nucleotide sequence”, “polynucleotide” or “nucleic acid” can be used interchangeably and are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g., RNA molecules).

The range of percent identity, between 20.00% and 99.99%, is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 20.00% and, up to, including 99.99%. These percentages are purely statistical and differences between two nucleic acid sequences can be distributed randomly and over the entire sequence length. For example, homologous sequences can exhibit a percent identity of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent with the sequences of the instant invention. Typically, the percent identity is calculated with reference to the full length, native, and/or naturally occurring polynucleotide. The terms “identical” or percent “identity”, in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

Both protein and nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson et al., 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; Gish et al., 1993). Sequence comparisons are, typically, conducted using default parameters provided by the vendor or using those parameters set forth in the above-identified references, which are hereby incorporated by reference in their entireties.

A “complementary” polynucleotide sequence, as used herein, generally refers to a sequence arising from the hydrogen bonding between a particular purine and a particular pyrimidine in double-stranded nucleic acid molecules (DNA-DNA, DNA-RNA, or RNA-RNA). The major specific pairings are guanine with cytosine and adenine with thymine or uracil. A “complementary” polynucleotide sequence may also be referred to as an “antisense” polynucleotide sequence or an “antisense sequence”.

Sequence homology and sequence identity can also be determined by hybridization studies under high stringency, intermediate stringency, and/or low stringency. Various degrees of stringency of hybridization can be employed. The more severe the conditions are, the greater the complementarity that is required for duplex formation. Severity of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under low, intermediate, or high stringency conditions by techniques well known in the art, as described, for example, in Keller and Manak (1987).

For example, hybridization of immobilized DNA on Southern blots with 32 P-labeled gene-specific probes can be performed by standard methods (Maniatis et al., 1982). In general, hybridization and subsequent washes can be carried out under intermediate to high stringency conditions that allow for detection of target sequences with homology to the exemplified polynucleotide sequence. For double-stranded DNA gene probes, hybridization can be carried out overnight at 20-25° C. below the melting temperature (T m ) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz et al., 1983).

Tm= 81.5° C.+16.6 Log [Na + ]+0.41 (% G+C )−0.61 (% formamide)−600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash);

(2) once at T m −20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (intermediate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnight at 10-20° C. below the melting temperature (T m ) of the hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. T m for oligonucleotide probes can be determined by the following formula:

T m (° C.)=2(number T/A base pairs) + 4(number G/C base pairs) (Suggs et al., 1981).

Washes can be carried out as follows:

(1) twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (low stringency wash);

(2) once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (intermediate stringency wash).

In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment >70 or so bases in length, the following conditions can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or 2×SSPE, 42° C.

Intermediate: 0.2× or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

By way of another non-limiting example, procedures using conditions of high stringency can also be performed as follows: Pre-hybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in pre-hybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10 6 cpm of 32 P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al. (1989) and Ausubel et al. (1989) are incorporated herein in their entirety.

Another non-limiting example of procedures using conditions of intermediate stringency are as follows: Filters containing DNA are pre-hybridized, and then hybridized at a temperature of 60° C. in the presence of a 5×SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2×SSC at 50° C. and the hybridized probes are detectable by autoradiography. Other conditions of intermediate stringency which may be used are well known in the art and as cited in Sambrook et al. (1989) and Ausubel et al. (1989) are incorporated herein in their entirety.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.

It is also well known in the art that restriction enzymes can be used to obtain functional fragments of the subject DNA sequences. For example, Bal31 exonuclease can be conveniently used for time-controlled limited digestion of DNA (commonly referred to as “erase-a-base” procedures). See, for example, Maniatis et al. (1982); Wei et al. (1983).

The present invention further comprises fragments of the polynucleotide sequences of the instant invention. Representative fragments of the polynucleotide sequences according to the invention will be understood to mean any nucleotide fragment having at least 5 successive nucleotides, preferably at least 12 successive nucleotides, and still more preferably at least 15, 18, or at least 20 successive nucleotides of the sequence from which it is derived. The upper limit for fragments as set forth herein is the total number of nucleotides found in the full-length sequence encoding a particular polypeptide (e.g., a polypeptide such as that of SEQ ID NO: 5).

In some embodiments, the subject invention includes those fragments capable of hybridizing under various conditions of stringency conditions (e.g., high or intermediate or low stringency) with a nucleotide sequence according to the invention; fragments that hybridize with a nucleotide sequence of the subject invention can be, optionally, labeled as set forth below.

The subject invention provides, in one embodiment, methods for the identification of the presence of nucleic acids according to the subject invention in transformed host cells or in cells isolated from an individual suspected of being infected by West Nile virus. In these varied embodiments, the invention provides for the detection of nucleic acids in a sample (obtained from the individual or from a cell culture) comprising contacting a sample with a nucleic acid (polynucleotide) of the subject invention (such as an RNA, mRNA, DNA, cDNA, or other nucleic acid). In a preferred embodiment, the polynucleotide is a probe that is, optionally, labeled and used in the detection system. Many methods for detection of nucleic acids exist and any suitable method for detection is encompassed by the instant invention. Typical assay formats utilizing nucleic acid hybridization includes, and are not limited to, 1) nuclear run-on assay, 2) slot blot assay, 3) northern blot assay (Alwine et al., 1977, 4) magnetic particle separation, 5) nucleic acid or DNA chips, 6) reverse Northern blot assay, 7) dot blot assay, 8) in situ hybridization, 9) kNase protection assay (Melton et al., 1984) and as described in the 1998 catalog of Ambion, Inc., Austin, Tex., 10) ligase chain reaction, 11) polymerase chain reaction (PCR), 12) reverse transcriptase (RT)-PCR (Berchtold, 1989), 13) differential display RT-PCR (DDRT-PCR) or other suitable combinations of techniques and assays. Labels suitable for use in these detection methodologies include, and are not limited to 1) radioactive labels, 2) enzyme labels, 3) chemiluminescent labels, 4) fluorescent labels, 5) magnetic labels, or other suitable labels, including those set forth below. These methodologies and labels are well known in the art and widely available to the skilled artisan. Likewise, methods of incorporating labels into the nucleic acids are also well known to the skilled artisan.

Thus, the subject invention also provides detection probes (e.g., fragments of the disclosed polynucleotide sequences) for hybridization with a target sequence or the amplicon generated from the target sequence. Such a detection probe will comprise a contiguous/consecutive span of at least 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. Labeled probes or primers are labeled with a radioactive compound or with another type of label as set forth above (e.g., 1) radioactive labels, 2) enzyme labels, 3) chemiluminescent labels, 4) fluorescent labels, or 5) magnetic labels). Alternatively, non-labeled nucleotide sequences may be used directly as probes or primers; however, the sequences are generally labeled with a radioactive element ( 32 P, 35 S, 3 H, 121 I) or with a molecule such as biotin, acetylaminofluorene, digoxigenin, 5-bromo-deoxyuridine, or fluorescein to provide probes that can be used in numerous applications.

Polynucleotides of the subject invention can also be used for the qualitative and quantitative analysis of gene expression using arrays or polynucleotides that are attached to a solid support. As used herein, the term array means a one-, two-, or multi-dimensional arrangement of full length polynucleotides or polynucleotides of sufficient length to permit specific detection of gene expression. Preferably, the fragments are at least 15 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. More preferably, the fragments are more than 100 nucleotides in length. In some embodiments the fragments may be more than 500 nucleotides in length.

For example, quantitative analysis of gene expression may be performed with full-length polynucleotides of the subject invention, or fragments thereof, in a complementary DNA microarray as described by Schena et al. (1995, 1996). Polynucleotides, or fragments thereof, are amplified by PCR and arrayed onto silylated microscope slides. Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution. The arrays are submerged in water for 2 min at 95° C., transferred into 0.2% SDS for 1 min, rinsed twice with water, air dried and stored in the dark at 25° C. mRNA is isolated from a biological sample and probes are prepared by a single round of reverse transcription. Probes are hybridized to 1 cm 2 microarrays under a 14×14 mm glass coverslip for 6-12 hours at 60° C. Arrays are washed for 5 min at 25° C. in low stringency wash buffer (1×SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1×SSC/0.2% SDS). Arrays are scanned in 0.1×SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.

Quantitative analysis of the polynucleotides present in a biological sample can also be performed in complementary DNA arrays as described by Pietu et al. (1996). The polynucleotides of the invention, or fragments thereof, are PCR amplified and spotted on membranes. Then, mRNAs originating from biological samples derived from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.

Alternatively, the polynucleotide sequences of the invention may also be used in analytical systems, such as DNA chips. DNA chips and their uses are well known in the art (see for example, U.S. Pat. Nos. 5,561,071; 5,753,439; 6,214,545; Schena 1996; Bianchi et al., 1997; each of which is hereby incorporated by reference in their entireties) and/or are provided by commercial vendors such as Affymetrix, Inc. (Santa Clara, Calif.). In addition, the nucleic acid sequences of the subject invention can be used as molecular weight markers in nucleic acid analysis procedures.

The subject invention also provides compositions of matter that comprise:

a) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15 or encoding one or more polypeptide fragment of SEQ ID NOs: 5, 9, 11, 13 or 15 as set forth in Table 9, 10, 11, 12, 15, or 16. In various aspects of the invention, these polynucleotides can have a G+C content of at least 40% and less than 50% or a G+C content as set forth in Table 13;

b) a polynucleotide sequence that is at least 70% (or a percentage as specified in the Table 14) identical to SEQ ID NO: 1, encodes a polypeptide comprising SEQ ID NO: 2 and has a G+C content of between about 40% and about 50% (or a specific G+C content as specified in Table 13);

c) a polynucleotide sequence at least 8 consecutive nucleotides of a polynucleotide sequence as set forth in (a) or (b);

d) a polynucleotide sequence comprising SEQ ID NO: 3, 4, 6, 7, 8, 10, or 12 or a fragment of at least 8 consecutive nucleotides of SEQ ID NO: 3, 4, 6, 7, 8, 10, or 12;

e) a polynucleotide that is complementary to the polynucleotides set forth in (a), (b), (c), or (d);

f) a polynucleotide that hybridizes under low, intermediate or high stringency with a polynucleotide sequence as set forth in (a), (b), (c), (d) or (e);

g) a genetic construct comprising a polynucleotide sequence as set forth in (a), (b), (c), (d) or (e);

h) a vector comprising a polynucleotide or genetic construct as set forth in (a), (b), (c), (d), (e), (f) or (g);

i) a host cell comprising a vector as set forth in (h), a genetic construct as set forth in (g), or a polynucleotide as set forth in any one of (a), (b), (c), (d) or (e);

j) a transgenic plant, plant cell, or plant part comprising a vector as set forth in (h), a genetic construct as set forth in (g) or a polynucleotide as set forth in any one of (a), (b), (c), (d) or (e); or

k) a probe comprising a polynucleotide according to (a), (b), (c), (d), (e) or (f) and, optionally, a label or marker.

The subject invention also provides genetic constructs comprising: a) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15, or a fragment thereof; b) a polynucleotide sequence having at least about 20% to 99.99% identity to a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15, or a fragment of SEQ ID NO: 5, 9, 11, 13 or 15, wherein said polypeptide has at least one of the biological activities of a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15, or a fragment thereof; c) a polynucleotide sequence encoding a polypeptide having at least about 20% to 99.99% identity to a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15, or a fragment of SEQ ID NO: 5, 9, 11, 13 or 15, wherein said polypeptide has at least one of the biological activities of a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15, or a fragment thereof; d) a polynucleotide sequence encoding a fragment of a polypeptide comprising SEQ ID NO: 5, 9, 11, 13 or 15, wherein said fragment has at least one of the activities of the polypeptide of SEQ ID NO: 5, 9, 11, 13 or 15; e) a polynucleotide sequence comprising SEQ ID NO: 3, 4, 6, 7, 8, 10, 12, or 14; f) a polynucleotide sequence having at least about 20% to 99.99% identity to the polynucleotide sequence of SEQ ID NO: 3, 4, 6, 7, 8, 10, 12, or 14; g) a polynucleotide sequence encoding multimeric construct; or h) a polynucleotide that is complementary to the polynucleotides set forth in (a), (b), (c), (d), (e), (f), or (g). Genetic constructs of the subject invention can also contain additional regulatory elements such as promoters and enhancers and, optionally, selectable markers.

Also within the scope of the subject instant invention are vectors or expression cassettes containing genetic constructs as set forth herein or polynucleotides encoding the polypeptides, set forth supra, operably linked to regulatory elements. The vectors and expression cassettes may contain additional transcriptional control sequences as well. The vectors and expression cassettes may further comprise selectable markers. The expression cassette may contain at least one additional gene, operably linked to control elements, to be co-transformed into the organism. Alternatively, the additional gene(s) and control element(s) can be provided on multiple expression cassettes. Such expression cassettes are provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette(s) may additionally contain selectable marker genes operably linked to control elements.

The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the invention, and transcriptional and translational termination regions. The transcriptional initiation region, the promoter, may be native or analogous, or foreign or heterologous, to the host cell. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.

Another aspect of the invention provides vectors for the cloning and/or the expression of a polynucleotide sequence taught herein. Vectors of this invention, including vaccine vectors, can also comprise elements necessary to allow the expression and/or the secretion of the said nucleotide sequences in a given host cell. The vector can contain a promoter, signals for initiation and for termination of translation, as well as appropriate regions for regulation of transcription. In certain embodiments, the vectors can be stably maintained in the host cell and can, optionally, contain signal sequences directing the secretion of translated protein. These different elements are chosen according to the host cell used. Vectors can integrate into the host genome or, optionally, be autonomously-replicating vectors.

The subject invention also provides for the expression of a polypeptide or peptide fragment encoded by a polynucleotide sequence disclosed herein comprising the culture of a host cell transformed with a polynucleotide of the subject invention under conditions that allow for the expression of the polypeptide and, optionally, recovering the expressed polypeptide.

The disclosed polynucleotide sequences can also be regulated by a second nucleic acid sequence so that the protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a protein or peptide may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression include, but are not limited to, the CMV-IE promoter, the SV40 early promoter region (Benoist and Chambon 1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980), the herpes simplex thymidine kinase promoter (Wagner et al., 1981), the regulatory sequences of the metallothionein gene (Brinster et al., 1982); prokaryotic vectors containing promoters such as the β-lactamase promoter (Villa-Kamaroff et al., 1978), or the tac promoter (deBoer et al., 1983); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., 1983) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984); promoter elements from yeast or fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, and/or the alkaline phosphatase promoter.

The vectors according to the invention are, for example, vectors of plasmid or viral origin. In a specific embodiment, a vector is used that comprises a promoter operably linked to a protein or peptide-encoding nucleic acid sequence contained within the disclosed polynucleotide sequences, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Expression vectors comprise regulatory sequences that control gene expression, including gene expression in a desired host cell. Exemplary vectors for the expression of the polypeptides of the invention include the pET-type plasmid vectors (Promega) or pBAD plasmid vectors (Invitrogen) or those provided in the examples below. Furthermore, the vectors according to the invention are useful for transforming host cells so as to clone or express the polynucleotide sequences of the invention.

The invention also encompasses the host cells transformed by a vector according to the invention. These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the polynucleotide sequences of the subject invention.

The host cell may be chosen from eukaryotic or prokaryotic systems, such as for example bacterial cells, (Gram negative or Gram positive), yeast cells (for example, Saccharomyces cereviseae or Pichia pastoris ), animal cells (such as Chinese hamster ovary (CHO) cells), plant cells, and/or insect cells using baculovirus vectors. In some embodiments, the host cells for expression of the polypeptides include, and are not limited to, those taught in U.S. Pat. Nos. 6,319,691, 6,277,375, 5,643,570, or 5,565,335, each of which is incorporated by reference in its entirety, including all references cited within each respective patent.

Furthermore, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure “native” glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to different extents.

Also provided are transformed plant cells, transgenic seeds, transgenic plant parts and transgenic plants which contain one or more polynucleotide sequence, genetic construct, vector, or expression cassette comprising one or more of the polynucleotides disclosed herein, or biologically active fragments thereof, operably linked to control elements. As used herein, the term “plant” includes algae and higher plants (including, but not limited to trees). Thus, algae, monocots, and dicots may be transformed with genetic constructs of the invention, expression cassettes, or vectors according to the invention. In certain preferred embodiments, tobacco plants or tobacco cell lines are transformed with genetic constructs according to the subject invention.

Thus, polypeptides useful in the production of the compositions or immunization protocols discussed in this application can be derived or obtained from a transgenic plant cell that has been genetically engineered to express a polypeptide comprising (consisting essentially of or consisting of) SEQ ID NO: 5, 9, 11, 13, 15, or fragments thereof. See, for example, U.S. Patent Pub. No: 2004/0268442 A1, the disclosure of which is hereby incorporated by reference in its entirety.

Transgenic plant is herein defined as a plant cell culture, plant cell line, plant tissue culture, lower plant, monocot plant, dicot plant, or progeny or part thereof derived from a transformed plant cell or protoplast, wherein the genome of the transformed plant contains foreign DNA, introduced by laboratory techniques, not originally present in a native, non-transgenic plant cell of the same species. The terms “transgenic plant” and “transformed plant” have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA molecule. Where appropriate, the polynucleotides encoding the polypeptides set forth herein can be optimized for expression in the transformed plants, plant cells or plant parts. That is, the genes can be synthesized using species-preferred codons corresponding to the species of interest. Methods are available in the art for synthesizing for example, plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989), herein incorporated by reference.

Construction of gene cassettes for expressing polypeptides in plants is readily accomplished utilizing well known methods, such as those disclosed in Sambrook et al. (1989); and Ausubel et al. (1987).

In preparing the constructs of this invention, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Adapters or linkers may be employed for joining the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.

In carrying out the various steps, cloning is employed, so as to amplify a vector containing the promoter/gene of interest for subsequent introduction into the desired host cells. A wide variety of cloning vectors are available, where the cloning vector includes a replication system functional in Escherichia coli ( E. coli ) and a marker which allows for selection of the transformed cells. Illustrative vectors include pBR322, pUC series, pACYC184, Bluescript series (Stratagene) etc. Thus, the sequence may be inserted into the vector at an appropriate restriction site(s), the resulting plasmid used to transform the E. coli host (e.g., E. coli strains HB101, JM101 and DH5α), the E. coli grown in an appropriate nutrient medium and the cells harvested and lysed and the plasmid recovered. Analysis may involve sequence analysis, restriction analysis, electrophoresis, or the like. After each manipulation, the DNA sequence to be used in the final construct may be restricted and joined to the next sequence, where each of the partial constructs may be cloned in the same or different plasmids.

Vectors are available or can be readily prepared for transformation of plant cells. In general, plasmid or viral vectors should contain all the DNA control sequences necessary for both maintenance and expression of a heterologous DNA sequence in a given host. Such control sequences generally include a leader sequence and a DNA sequence coding for translation start-signal codon, a translation terminator codon, and a DNA sequence coding for a 3′ UTR signal controlling messenger RNA processing. Selection of appropriate elements to optimize expression in any particular species is a matter of ordinary skill in the art utilizing the teachings of this disclosure. Finally, the vectors should desirably have a marker gene that is capable of providing a phenotypical property which allows for identification of host cells containing the vector.

The activity of the foreign coding sequence inserted into plant cells is dependent upon the influence of endogenous plant DNA adjacent the insert. Generally, the insertion of heterologous genes appears to be random using any transformation technique; however, technology exists for producing plants with site specific recombination of DNA into plant cells (see WO 91/09957). Any method or combination of methods resulting in the expression of the desired sequence or sequences under the control of the promoter is acceptable.

The present invention is not limited to any particular method for transforming plant cells. Technology for introducing DNA into plant cells is well-known to those of skill in the art. Four basic methods for delivering foreign DNA into plant cells have been described. Chemical methods (Graham and van der Eb, 1973; Zatloukal et al., 1992); physical methods including microinjection (Capecchi, 1980), electroporation (Wong and Neumann 1982; Fromm et al., 1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993); viral methods (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson 1988; Eglitis et al., 1988); and receptor-mediated methods (Curiel et al., 1991; Curiel et al., 1992; Wagner et al., 1992).

The introduction of DNA into plant cells by means of electroporation is well-known to those of skill in the art. Plant cell wall-degrading enzymes, such as pectin-degrading enzymes, are used to render the recipient cells more susceptible to transformation by electroporation than untreated cells. To effect transformation by electroporation one may employ either friable tissues such as a suspension culture of cells, or embryogenic callus, or immature embryos or other organized tissues directly. It is generally necessary to partially degrade the cell walls of the target plant material with pectin-degrading enzymes or mechanically wounding in a controlled manner. Such treated plant material is ready to receive foreign DNA by electroporation.

Another method for delivering foreign transforming DNA to plant cells is by microprojectile bombardment. In this method, microparticles are coated with foreign DNA and delivered into cells by a propelling force. Such micro particles are typically made of tungsten, gold, platinum, and similar metals. An advantage of microprojectile bombardment is that neither the isolation of protoplasts (Cristou et al., 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen onto a filter surface covered with corn cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids.

Agrobacterium -mediated transfer is a widely applicable system for introducing foreign DNA into plant cells because the DNA can be introduced into whole plant tissues, eliminating the need to regenerate an intact plant from a protoplast. The use of Agrobacterium -mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described in Fraley et al. (1985) and Rogers et al. (1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described in Spielmann et al. (1986) and Jorgensen et al. (1987).

Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium , allowing for convenient manipulations. Moreover, recent technological advances in vectors for Agrobacterium -mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various proteins or polypeptides. Convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations.

Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., 1985; Marcotte et al., 1988). Application of these systems to different plant species depends on the ability to regenerate the particular species from protoplasts.

Once the plant cells have been transformed, selected and checked for antigen expression, it is possible in some cases to regenerate whole fertile plants. This will greatly depend on the plant species chosen. Methods for regenerating numerous plant species have been reported in the literature and are well known to the skilled artisan. For practice of the present invention, it is preferable to transform plant cell lines that can be cultured and scaled-up rapidly by avoiding the generally lengthy regeneration step. In addition, the use of plant cell cultures avoids open field production and greatly reduces the chances of gene escape and food contamination. Tobacco suspension cell cultures such as NT-1 and BY-2 (An, 1985) are preferred because these lines are particularly susceptible to handling in culture, are readily transformed, produce stably integrated events and are amenable to cryopreservation.

The tobacco suspension cell line, NT-1, is suitable for the practice of the present invention. NT-1 cells were originally developed from Nicotiana tabacum L.cv. bright yellow 2. The NT-1 cell line is widely used and readily available; though, any tobacco suspension cell line is consistent with the practice of the invention. NT-1 cells suitable for use in the examples below are available from the American Type Culture Collection under accession number ATCC No. 74840. See also U.S. Pat. No. 6,140,075, herein incorporated by reference in its entirety.

Many plant cell culture techniques and systems ranging from laboratory-scale shaker flasks to multi-thousand liter bioreactor vessels have been described and are well know in the art of plant cell culture. See for example Fischer, R. et al (1999) and Doran, P. (2000). After the transformed plant cells have been cultured to the mass desired, they are harvested, gently washed and placed in a suitable buffer for disruption. Many different buffers are compatible with the present invention. In general the buffer is an aqueous isotonic buffered salt solution at or near a neutral pH value, with or without detergent to solubilize membrane-bound proteins. Preferred buffers include Dulbecco's Phosphate Buffered Saline, PBS containing 1 mM EDTA, and MOPS (3-(N-Morpholino)propanesulfonic acid).

In one embodiment, cells can be disrupted by sonication. The washed cells are placed in buffer in a range of about 0.01 mg/ml to about 5.0 mg/ml, preferably in a range of about 0.1 mg/ml to about 0.5 mg/ml (washed wet weight cells per volume of buffer). Many commercially available sonication instruments are consistent with the invention and sonication times range from about 5 to about 20 seconds, preferably about 15 to about 20 seconds. The resulting cell fragments may range in size from a few microns to several hundred microns and expose the polypeptide or immunogenic fragments thereof.

The subject invention also concerns DNA vaccine compositions that can be employed to elicit an immune response or a protective immune response. In this aspect of the invention, an amount of a composition comprising recombinant DNA or mRNA encoding a polypeptide as provided herein (or a fragment thereof) is administered to an individual in an amount sufficient to elicit an immune response or protective immune response in said individual. Signal sequences may be deleted from the nucleic acid encoding an antigen of interest and the individual may be monitored for the induction of an immune response according to methods known in the art. A “protective immune response” or “therapeutic immune response” refers to a CTL (or CD8 + T cell), an HTL (or CD4 + T cell), and/or a protective humoral immune response to an antigen that, in some way, prevents or at least partially arrests disease symptoms, side effects or progression. In the context of this invention, such a protective or therapeutic response provides increased survival rates (reduced mortality) in immunized individuals as compared to non-immunized individuals or a reduction in viral shedding in immunized individuals challenged with West Nile virus.

In another embodiment, the subject invention further comprises the administration of polynucleotide (DNA) vaccines in conjunction with a polypeptide antigen, or composition thereof, of the invention. In a preferred embodiment, the antigen is the polypeptide that is encoded by the polynucleotide administered as the polynucleotide vaccine. As a particularly preferred embodiment, the polypeptide antigen is administered as a booster subsequent to the initial administration of the polynucleotide vaccine.

A further embodiment of the subject invention provides for the induction of an immune response to the novel West Nile virus antigens disclosed herein (see, for example, the polypeptides and peptide fragments set forth herein) using a “prime-boost” vaccination regimen known to those skilled in the art. In this aspect of the invention, a DNA vaccine or polypeptide antigen of the subject invention is administered to an individual in an amount sufficient to “prime” the immune response of the individual. The immune response of the individual is then “boosted” via the administration of: 1) one or a combination of: a peptide, polypeptide, and/or full length polypeptide antigen of the subject invention (optionally in conjunction with a immunostimulatory molecule and/or an adjuvant); or 2) a viral vector that contains nucleic acid encoding one, or more, of the same or, optionally, different, antigen constructs, and/or peptide antigens set forth herein. In some alternative embodiments of the invention, a gene encoding an immunostimulatory molecule may be incorporated into the viral vector used to “boost the immune response of the individual. Exemplary immunostimulatory molecules include, and are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-15, 11-16, 11-18, IL-23, IL-24, erythropoietin, G-CSF, M-CSF, platelet derived growth factor (PDGF), MSF, FLT-3 ligand, EGF, fibroblast growth factor (FGF; e.g., aFGF (FGF-1), bFGF (FGF-2), FGF-3, FGF-4, FGF-5, FGF-6, or FGF-7), insulin-like growth factors (e.g., IGF-1, IGF-2); vascular endothelial growth factor (VEGF); interferons (e.g., IFN-γ, IFN-α, IFN-β); leukemia inhibitory factor (LIF); ciliary neurotrophic factor (CNTF); oncostatin M; stem cell factor (SCF); transforming growth factors (e.g., TGF-α, TGF-β1, TGF-β2, TGF-β3), or chemokines (such as, but not limited to, BCA-1/BLC-1, BRAK/Kec, CXCL16, CXCR3, ENA-78/LIX, Eotaxin-1, Eotaxin-2/MPIF-2, Exodus-2/SLC, Fractalkine/Neurotactin, GROalpha/MGSA, HCC-1, I-TAC, Lymphotactin/ATAC/SCM, MCP-1/MCAF, MCP-3, MCP-4, MDC/STCP-1, ABCD-1, MIP-1α, MIP-1β, MIP-2α/GROβ, MIP-3α/Exodus/LARC, MIP-3β/Exodus-3/ELC, MIP-4/PARC/DC-CK1, PF-4, RANTES, SDF1α, TARC, or TECK). Genes encoding these immunostimulatory molecules are known to those skilled in the art and coding sequences may be obtained from a variety of sources, including various patents databases, publicly available databases (such as the nucleic acid and protein databases found at the National Library of Medicine or the European Molecular Biology Laboratory), the scientific literature, or scientific literature cited in catalogs produced by companies such as Genzyme, Inc., R&D Systems, Inc, or InvivoGen, Inc. [see, for example, the 1995 Cytokine Research Products catalog, Genzyme Diagnostics, Genzyme Corporation, Cambridge Mass.; 2002 or 1995 Catalog of R&D Systems, Inc (Minneapolis, Minn.); or 2002 Catalog of InvivoGen, Inc (San Diego, Calif.) each of which is incorporated by reference in its entirety, including all references cited therein].

Methods of introducing DNA vaccines into individuals are well-known to the skilled artisan. For example, DNA can be injected into skeletal muscle or other somatic tissues (e.g., intramuscular injection). Cationic liposomes or biolistic devices, such as a gene gun, can be used to deliver DNA vaccines. Alternatively, iontophoresis and other means for transdermal transmission can be used for the introduction of DNA vaccines into an individual.

Viral vectors for use in the subject invention can have a portion of the viral genome deleted to introduce new genes without destroying infectivity of the virus. The viral vector of the present invention is, typically, a non-pathogenic virus. At the option of the practitioner, the viral vector can be selected so as to infect a specific cell type, such as professional antigen presenting cells (e.g., macrophage or dendritic cells). Alternatively, a viral vector can be selected that is able to infect any cell in the individual. Exemplary viral vectors suitable for use in the present invention include, but are not limited to poxvirus such as vaccinia virus, avipox virus, fowlpox virus, a highly attenuated vaccinia virus (such as Ankara or MVA [Modified Vaccinia Ankara]), retrovirus, adenovirus, baculovirus and the like. In a preferred embodiment, the viral vector is Ankara or MVA.

General strategies for construction of vaccinia virus expression vectors are known in the art [see, for example, Smith and Moss, 1984; U.S. Pat. No. 4,738,846 (hereby incorporated by reference in its entirety)]. Sutter and Moss (1992) and Sutter et al. (1994) disclose the construction and use as a vector, a non-replicating recombinant Ankara virus (MVA) which can be used as a viral vector in the present invention.

Compositions comprising the subject polynucleotides can include appropriate nucleic acid vaccine vectors (plasmids), which are commercially available (e.g., Vical, San Diego, Calif.) or other nucleic acid vectors (plasmids), which are also commercially available (e.g., Valenti, Burlingame, Calif.). Alternatively, compositions comprising viral vectors and polynucleotides according to the subject invention are provided by the subject invention. In addition, the compositions can include a pharmaceutically acceptable carrier, e.g., saline. The pharmaceutically acceptable carriers are well known in the art and also are commercially available. For example, such acceptable carriers are described in E. W. Martin's Remington's Pharmaceutical Science , Mack Publishing Company, Easton, Pa.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

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