US 20090069256A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0069256A1 Smith et al. (43) Pub. Date: Mar. 12, 2009

(54) ENHANCING PROTEIN EXPRESSION Related U.S. Application Data (60) Provisional application No. 60/576,819, filed on Jun. (76) Inventors: Larry R. Smith, San Diego, CA 4, 2004. (US); Vafa Shahabi, Valley Forge, PA (US); Maninder K. Sidhu, New Publication Classification City, NY (US) (51) Int. Cl. A63L/7088 (2006.01) Correspondence Address: CI2N IS/II (2006.01) HUNTON & WILLIAMS LLP CI2N 15/87 (2006.01) INTELLECTUAL PROPERTY DEPARTMENT A6IP 43/00 (2006.01) 1900 KSTREET, N.W., SUITE 1200 CI2N IS/00 (2006.01) WASHINGTON, DC 20006-1109 (US) (52) U.S. Cl...... 514/44; 536/23.1; 536/23.53: 536/23.5; 536/23.6:536/23.7:536/23.72:536/23.74; 435/455; 435/320.1 (21) Appl. No.: 11/628,455 (57) ABSTRACT (22) PCT Fled: Jun. 6, 2005 Modified polynucleotide compositions providing enhanced gene expression and methods for preparing said compositions (86) PCT NO.: PCT/US05/19592 are disclosed. Methods of using the compositions, such as in screening assays, diagnostic tools, kits, etc. and for preven S371 (c)(1), tion and/or treatment of diseases and disorders are also dis (2), (4) Date: Nov. 8, 2007 closed.

SWall (3733) DraI(3734) Stul (52) BclI (3512)

ASCI (3496) ApaI (3482) BSInI (3370) BglII (457) 2->BGHpolyA BspEI (3271) BspMI (540) BseRI (3241) human IL-15 (BH15) Psp1406I (667) BamHI (3071) “HuigE leader

PflMI (3056) kanamycin Eael (768) NsiI (791) (3737 bp) XmnI (806) BpmI (2976)1 AgeI (846) MsiI (2745) BSrFI (846) SnaBI (2722) Eam1105I (920) BSaAI (2722) DraII (1155) Asel (2390) Spel (2383) EcoRV (2291) ClaI (2285) NruI (2254) BSrBI (1965) AfilII (1896) MunI (2209) Patent Application Publication Mar. 12, 2009 Sheet 1 of 14 US 2009/006925.6 A1

s

d co Co CD CD CD Co. Co. dAs as 3 N Co Ltd cro N. P. (S3LKOON3ldSoO 10.Si.) AINOWOdSEAN 09 NU0 Patent Application Publication Mar. 12, 2009 Sheet 2 of 14 US 2009/006925.6 A1

PmeI (4156) EcoRI (4167) BCI (94) Stu (55) BglII (458) PpuMI (4123) BspMI (550)

BstXI (3906) Psp1406I (669) BSmI (3902) - BGirolya Tsp45I (3760) BSal (3661) kanamycin

BSgI (3484)

PVuII (3418) HIV-1gagp37 WLV006 (4419 bp)

Sal I (3049) BSSHII (3043) BpmI (2998) HCMV promoter BSmBI (2974) Ori Seq Dral (1161)

NCOI (2745) SnaBI (2725) BsaNI (2725) Afi II (1897)

Ase (2392) EcoRV (2294) Muni (2210) BsrBI (1968) Nrul (2257)

FIG. 2 Patent Application Publication Mar. 12, 2009 Sheet 3 of 14 US 2009/006925.6 A1

AscI (4157) BSSHII (4157) SacI (4168)

kanamycin

SacI (3284)\ . WLWO Ola (4397 bp) SacI (3182) SacI (3098) NCoI (3059) BSSHII (3043) SalI (3049) (7braII (1161) SacI (2953)

NCOI (2745)

1842 (1841) BspLU11I (1897) EcoRV (2294)

FIG. 3 Patent Application Publication Mar. 12, 2009 Sheet 4 of 14 US 2009/006925.6 A1

ASCI (5630) Bsshi I (5630) junction marker (5628) EcoRI (5478)

kanamycin

env 6101, less preferred COdons

WLV118.gCS 1 (5870 bp)

HCMV promoter

ECORI (3468)

EcoRI (3483) Sal (3049) NCOI

junction marker (3060) BSSHII (3043)

F.G. 4 Patent Application Publication Mar. 12, 2009 Sheet 5 of 14 US 2009/006925.6 A1

ASCI (5107) BclI (5722) SphI (5935) junction marker (5946)

e

BGH Poly A NsiI (796)

Kanamycin & DraIII (1161). Econ) (4543)A • HIV-1 envgp160 from 6101 primary isolate

WLW119 R (5946 bp) Ori Sequence

Bsu36f(4223)N BSrBI (1968)

NruI (2257) HCMV P rOmoter ClaI (2287) DraIII (3510) EcoRV (2294) NsiI (3448) Spe (2461)

KpnI (3261) Sal (3126) BSmBI (3051) NcoI (2822)

FIG. 5 Patent Application Publication Mar. 12, 2009 Sheet 6 of 14 US 2009/006925.6 A1

RD CELS 25OOOOO 2. C: CURRENTCLINICAL CONSTRUCT 2000000 LP:MODIFIEDIL-15 2 O: OPTIMIZED ALTERNATIVE 15000 BH: OPTIMIZED ALTERNATIVE 2 S 1000000 500000 5000 as 52 5 5 5 g H H H H 5a , , , S2 a FIG.6A

COS7 CELLS 1800C 1600C C: CURRENTCLINICAL CONSTRUCT a LP:MODIFIEDIL-15 14000 O: OPTIMIZED ALTERNATIVE 2 1200c BH: OPTIMIZED ALTERNATIVE2 S 1000 is 800C ge 6000 400 2000

OOO 1 Lad a 1 4 1 S2 S , , , , is a 5 F G. 6 B Patent Application Publication Mar. 12, 2009 Sheet 7 of 14 US 2009/006925.6 A1

Hela CELS 5000 CCURRENTCNICALCONSTRUCT so-HERE : 3500 OOPTIMIZEDATERNATIVEBHOPTIMIZED ALTERNATIVE2 20 is a H e H 0. Y to 1 ha o1. 1 S2 i. i. i. i. 2 3, 2, 3, 2 E 5 = c F.G. 6C Patent Application Publication Mar. 12, 2009 Sheet 8 of 14 US 2009/006925.6 A1

1800.00 1600.00 RD - 1400.00 1200.00 100000 800.00 60000 40000 LP-15-IgE 200.00 BH-15-gE 0.00

FIG. 7A

9000.00 800000 - 700000 2 600000 500000 400000 is 300000 2000.00 100000 0.00 Patent Application Publication Mar. 12, 2009 Sheet 9 of 14 US 2009/006925.6 A1

9

OZ?E

EIN00.10N.9U Patent Application Publication Mar. 12, 2009 Sheet 10 of 14 US 2009/006925.6 A1

120.00 100.00 80.00 6000 4000 2000 OOO VECTOR CURRENTL15 LS-gELEADER 0-15-gELEADER LP15-gELEADER BHS-LEADER

FIG, 9 Patent Application Publication Mar. 12, 2009 Sheet 11 of 14 US 2009/0069256A1

DAY2 DAY5 40.00 35.00 O DAY9 3000 DAY.15 2500 o - 2000 3 1500 1000 i 5.00

0.00 CURRENTIL15 IL15-gELEADER 0-15-g LEADER LP15-IgELEADE

FIG 10 Patent Application Publication Mar. 12, 2009 Sheet 12 of 14 US 2009/0069256A1

ASCI (3496) XhoI (3489) Ava (3489) ApaI(3482) BsgI (3430) BSaBI (3435) Swal (3733) DraI (3734). StuI (52) MscI (3317) BstEII (3219) PvuII (3210) ne2. BcgI-1 (3164) BglII (457) BcgI-2 (3164) PpuMI (3149) $7,4 2. IL-15 (P 15) Psp1406I (667) EcoO109I (3149) HugE leader kanamycin NsiI (791) BamHI (3071) -AgeI (846) PflMI (3056) BSrFI (846) (3737 bp) BSmBI (2979) Eam1105I (920) HCMV IE promoter/untranslated region SnaBI (2722) BSaAI (2722) Asel (2390) SpeI (2383) EcoRV (2291) ClaI (2285) AflIII (1896) NruI (2254) MunI (2209) FIG 11 Patent Application Publication Mar. 12, 2009 Sheet 13 of 14 US 2009/006925.6 A1

Sphl (3721) Swal (3733) DraI (3734) StuI (52) ASCI (3496) ApaI(3482) 2

4. BGHpolyA BspMI (540) PshAI (3096) human IL-15 (LP15) Psp1406I (667) BamHI (3071) Eael (768) PflMI (3056) % Hu IgEleader kanalycin XmnI (806) BSIBI (2979) Age I (846)

SnaBI (2722) (3737 bp) BsrFI (846) BSaAI (2722) Eam1105I (920)

AseI (2390) DraIII (1155) Spel. (2383) EcoRV (2291) ClaI (2285) Munl (2209)

BsrBI (1965) FIG. 12 Patent Application Publication Mar. 12, 2009 Sheet 14 of 14 US 2009/006925.6 A1

Swal (3733) DraI(3734) StuI (52) BclI (3512)

ASCI (3496) Apal (3482) BSmI (3370) BglII (457) BspEI (3271) BSOMI

BseRI (3241) spMI (540) 4 human IL-15 (BH15) Psp1406I (667) BamHI (3071) HuigE leader

PflMI (3056-1 kanamycin Earl's BpmI (2976)1 (3737 bp) E: 888 MsiI (2745) A BsrFI (846)

E:Sa V Eam1105I (920) Dral.II (1155) Asel (2390) Spel. (2383) EcoRV (2291) Seas? Clai (2285) NruI (2254) BSrBI (1965) AfIII (1896) MunI (2209) FIG. 13 US 2009/006925.6 A1 Mar. 12, 2009

ENHANCING PROTEIN EXPRESSION B1) which limit the expression levels of gag by inhibiting nuclear export of these transcripts. FIELD OF THE INVENTION 0005 IL-15 exemplifies the problem inherent in poor gene expression. IL-15 is a pluripotent cytokine that is secreted by 0001. The present invention relates to polynucleotide antigen presenting cells Such as monocytes/macrophages and compositions that provide enhanced efficiency in the expres dendritic cells, but also a variety of nonlymphoid tissues. sion of proteins or polypeptides by genes in mammalian cells IL-15, in addition to being a growth and survival factor for (i.e., resulting in an increase in the levels of the proteins or memory CD8+ T cells, is also a potent activator of effector polypeptides encoded by the genes). Such as viral, bacterial memory CD8+ T cells, both in healthy and HIV-infected and mammalian genes, as well as methods for preparing said individuals. Because IL-15 is a prototypic Th1 cytokine, and compositions. In particular, the invention provides poly by virtue of its activity as a stimulator of T cells, NK cells, nucleotide sequences that provide enhanced gene expression LAK (lymphokine-activated killer) and TILs (tumor infiltrat over the corresponding wild-type polynucleotides. Also pro ing lymphocytes), IL-15 is a potential candidate for use as a vided are methods of using the polynucleotide compositions molecular adjuvantalong with HIV DNA vaccines to enhance in prevention and treatment of diseases and disorders (e.g., cellular immune responses. However, one major limiting fac immuno-therapeutic, immuno-prophylactic and genetic tor for its use as a genetic adjuvant, remains its poor expres therapy uses and the like), such as in DNA and RNA vaccines sion due to its complex regulation at the levels of mRNA (e.g., DNA vaccines for preventing/treating HIV/AIDS) as transcription and translation and, protein translocation and well as in biological assays, diagnostics and the like. secretion. 0006 Further, DNA vaccines, which are being studied for BACKGROUND OF THE INVENTION many diseases, including HIV, influenza, tuberculosis and malaria, usually work by injecting specially reproduced 0002 The level of protein expressed by a gene is crucial to genetic material of the organism directly into the body. This in vivo responses/effects involving the protein, as well as in genetic material encodes information that gets the individu vitro assays involving the protein. Under Some circumstances al’s own cells to make the vaccine. DNA vaccines have shown and for reasons not fully characterized, however, in vitro some impressive results in animals. Studies by Merck & Co. and/or in vivo benefits of the protein product of a gene are demonstrated that a DNA vaccine can prevent influenza in compromised because the gene is not adequately expressed in animals. cells. Poor protein expression is encountered in a number of 0007. In the area of HIV disease, DNA vaccines have different contexts. For example, poor expression of proteins generally not been able to stimulate strong immune responses by eukaryotic genes in prokaryotic cells has been previously in people. It has been suggested that DNA vaccines are less reported (see Seed et al., U.S. Pat. Nos. 5,786,464 and 5,795, effective in humans than in Smaller animals as a result of the 737). The poor expression of proteins by viral genes in mam problem of scaling up doses, where it is not practical to give malian cells has also been described (see Schwartz et al., J. large enough amounts of these vaccines to match the doses Virol. 66(12):7176-7182 (1992), Schneider et al., J. Virol., given to mice or monkeys. Interest in DNA vaccines either for 71(7):4892-4903 (1997) and Pavlakis et al., U.S. Pat. No. prevention or treatment is therefore likely to depend on find 6,414, 132 B1). However, the poor expression of certain viral, ing new and more efficient ways to present them to the bacterial and mammalian genes, in mammalian cells remains immune system. An approach that improves the expression of a significant problem from the standpoint of both in vivo uses a protein, Such as IL-15 for use as an adjuvant in a DNA of the protein products and in vitro uses in assays and the like. vaccine against HIV/AIDS, for example, is thus highly desir 0003. There are a number of factors that influence the able. levels of gene expression of proteins in mammalian cells and 0008 Various techniques have been proposed for optimiz that account for, or at least contribute to, the poor expression ing expression of genes, particularly for poorly expressed observed for certain genes in these cells. In some instances, genes. For example, one approach involved selectively translational mechanisms are responsible for the poor expres replacing wild-type codons encompassing inhibitory Sion. For example, it has been recognized that in certain sequences with other codons to eliminate the inhibitory wild-type genes, the naturally occurring nucleic acid effect. However, the sequence motifs that define either insta sequences of the genes are rich in adenine (A) and/or uracil bility or inhibitory sequences are not readily apparent and (U) (if the polynucleotide is RNA) or adenine (A) and/or therefore not easily identified. Several genes (e.g. E7 and En thymine (T) (if the polynucleotide is DNA) and biased toward among others) which appear to also contain inhibitory “disfavored codons'. The term "disfavored codons, as used sequences have not yet been mapped to identify the location herein, refers to codons that contain A, U, or T in the third of inhibitory sequences and there are no straightforward pre (“wobble') position of the codon nucleotide triplet. It has Scriptions from the gag work to predict how to eliminate been suggested in the art (see Haas et al., Current Biol. 6:315 inhibitory sequences from these genes. 324, 1996) that certain wild-type genes are not handled effi 0009 Further, a complete “codon optimized' version of ciently by the translational machinery of mammalian cells. gp120 envelope has been described (see Haas et al., Current 0004. Also, in addition to translational mechanisms Biology, 6:315-324, 1996; Andre et al., J. Virology, 72: 1497 accounting for poorly-expressed genes, there have been vari 1503) in which all “non-preferred wild-type codons from ous AU rich RNA instability sequences discovered in several env were replaced with “preferred codons and found to messenger RNAs (mRNAs) which do not directly impact the enhance expression levels. translatability of a given mRNA, but limit protein expression 0010 Previously available approaches, as described by increasing mRNA turnover. Further, several specific above, impose Stringent requirements in their application. In “inhibitory’ sequences contained within the HIV-1 gag ORF particular, these approaches require the use of "preferred have been described (see Pavlakis, U.S. Pat. No. 6,414,132 codons, or alternatively, identification of specific “inhibitory US 2009/006925.6 A1 Mar. 12, 2009 sequences.” For example, the technology described by Seed 0016. According to an embodiment, the present invention requires incorporation of “preferred codons' and purportedly provides a method of preventing or treating a disease in a depends on invoking the translational enhancement as the mammal comprising: administering to the mammal an effec mechanism of increased protein levels. tive amount of one or more compositions of the invention. 0011) “Preferred codons,” as defined by Seed, are GCC for 0017. According to a further embodiment, the present Ala, CGC for Arg, AAC for Asn., GAC for Asp, TGC for Cys, invention provides a method for enhancing expression of a CAG for Gln, GGC for Gly, CAC for His, ATC for lie, CTG gene comprising: expressing in vivo or in vitro a modified for Leu, AAG for Lys, CCC for Pro, TTC for Phe, AGC for polynucleotide of the invention. Ser, ACC for Thr, TAC for Tyr, and GTG for Val. According to 0018. According to another embodiment, the present Seed, “less preferred codons” are GGG for Gly, ATT for lie, invention provides a method for preparing a polynucleotide CTC for Leu, TCC for Ser, and GTC for Val. Seed also teaches that provides enhanced expression of a gene comprising: that all codons which do not fit the description of preferred assembling oligonucleotides comprising Surrogate codons to codons or less preferred condons are “non-preferred codons.” form a modified polynucleotide comprising a predetermined 0012. Accordingly, Seed's approach demands the use of nucleic acid sequence wherein the nucleotides cytosine (C) or the one specific codon prescribed in each instance and the guanine (G) occupy the wobble position of each of said Sur replacement of every codon or nearly every codon in a rogate codons in place of the corresponding nucleotides Sequence. adenine (A), uracil (U) or thymine (T) of a naturally-occur 0013 Likewise, the technology described by Pavlakis ring polynucleotide that expresses the same protein or requires identification of inhibitory/instability sequences and polypeptide as said modified polynucleotide. the alteration of those specifically identified inhibitory/insta 0019. According to yet another embodiment, the present bility sequences. According to Pavlakis, an inhibitory/insta invention provides a method for preparing a polynucleotide bility sequence of a transcript is a regulatory sequence that that provides enhanced expression of a gene comprising: (1) resides within an mRNA transcript and is either (1) respon determining for said gene a modified nucleic acid sequence sible for rapid turnover of that mRNA and can destabilize a comprising Surrogate codons in which the nucleotides second indicator/reporter mRNA when fused to that indica cytosine (C) or guanine (G) occupy the wobble position in tor/reporter mRNA, or is (2) responsible for underutilization place of the corresponding nucleotides adenine (A) or uracil of a mRNA and can cause decreased protein production from (U) or thymine (T) of a naturally-occurring polynucleotide a second indicator/reporter mRNA when fused to that second that expresses the same protein or polypeptide as said modi indicator/reporter mRNA or (3) both of the above. The pro fied polynucleotide; (2) selecting oligonucleotides having cedures to locate and mutate the inhibitory/instability nucleotide sequences corresponding to portions of said deter sequences are described in detail by Pavlakis. Accordingly, mined recombinant nucleic acid sequence; and (3) assem this approach is experimental result-dependent in that it bling the oligonucleotides to form a recombinant polynucle requires preliminary experimentation to identify specific otide comprising the determined recombinant nucleic acid regions of sequence for targeted mutation. Sequence. 0014 Polynucleotide compositions that provide enhanced 0020. According to a still further embodiment, the present gene expression while obviating any requirement to alter each invention provides a method for enhancing expression of a codon to a “preferred codon’ or identify “inhibitory gene comprising: altering a wild-type polynucleotide so that sequences’ provide certain benefits. These benefits include a naturally-occurring codon having adenine (A), uracil (U) or not only improved efficiency, cost-effectiveness, consistency thymine (T) in the wobble position is replaced by a surrogate and accuracy in improving the expression of certain genes, codon having cytosine (C) or guanine (G) in the wobble but also the ability to achieve a far greater Scope of applica position, said Surrogate codon encoding the same amino acid bility (i.e., the ability to attain Such improved gene expression as the naturally-occurring codon. possible for genes for which it was previously not possible (or 0021. According to another embodiment, the present at least highly inefficient) using previously available technol invention provides a modified polynucleotide comprising a ogy). It would be desirable to have an approach to attain nucleic acid sequence comprising Surrogate codons in which enhanced gene expression that avoids the stringent require the nucleotides cytosine (C) or guanine (G) occupy the ments of previous approaches. Accordingly, it would be desir wobble position in place of the corresponding nucleotides able to have an approach to attain enhanced gene expression adenine (A) or uracil (U), in RNA, or adenine (A) or thymine without having to alter all the codons of the gene to preferred (T), in DNA, of a naturally-occurring polynucleotide that codons or identify inhibitory sequences of the gene and then expresses the same protein or polypeptide as said modified altering those sequences. Moreover, it would be desirable to polynucleotide. have an approach that does not target, define, nor rely upon a 0022. According to a further embodiment, the present specific transcriptional or translational mechanism for invention provides a modified polynucleotide comprising a improved gene expression. nucleic acid sequence in which each codon encoding alanine is GCG, each codon encoding arginine is CGG or AGG, each SUMMARY OF THE INVENTION codon encoding leucine is CTC, each codon encoding proline is CCT or CCG, each codon encoding glutamic acid is GAG, 0015 The present invention provides enhanced gene each codon encoding glycine is GGG, each codon encoding expression in mammalian cells. In particular, the present isoleucine is ATT, each codon encoding serine is TCC, each invention provides modified polynucleotides with signifi codon encoding threonine is ACG, and each codon encoding cantly improved expression over their wild-type counter valine is GTC. parts. The present invention also provides compositions for 0023. According to still another embodiment, the present preventing and treating conditions, as well as compositions invention provides a modified polynucleotide comprising a for use in assays, vectors, diagnostic tools and the like. nucleic acid sequence having the general formula: —(X) - US 2009/006925.6 A1 Mar. 12, 2009

(Y), (X), , wherein X represents non-surrogate codons acid as the corresponding wild-type codons in the naturally having the nucleic acid sequence of any of the corresponding occurring polynucleotide that encodes the same protein or wild-type codons in the naturally-occurring polynucleotide polypeptide as said modified polynucleotide; whereini is any that encode the same protein or polypeptide as said recombi positive integer of at least 0; and wherein j is any positive nant polynucleotide, said wild-type codons having cytosine integer of at least 1. (C) or guanine (G) in the wobble position, wherein Y repre 0028. According to another embodiment, the present sents Surrogate codons having a nucleic acid sequence that is invention provides a composition comprising: (a) the nucleic different from the corresponding wild-type codons in the acid sequence of any of SEQ ID NOS: 1, 3 or 5; (b) an naturally-occurring polynucleotide that encode the same pro immunogenic encoding portion of (a); or (c) a nucleic acid tein or polypeptide as said recombinant polynucleotide, said sequence that hybridizes under Stringent conditions to any of wild-type codons having adenine (A) or uracil (U) or thymine (a) or (b). (T) in the wobble position, said Surrogate codons having 0029. According to a still further embodiment, the present cytosine (C), guanine (G) or thymine (T) in the wobble posi invention provides a composition comprising a polynucle tion and encoding the same amino acid as the corresponding otide comprising the nucleic acid sequence of any of SEQID wild-type codons in the naturally-occurring polypeptide that NOS: 1, 3 or 5; and a vector. encodes the same protein or polypeptide as said modified 0030. According to another embodiment, the present polynucleotide, whereini is any positive integer of at least 0: invention provides a composition comprising: a recombi and whereinj is any positive integer of at least 1. nantly expressed protein or polypeptide encoded by a modi 0024. According to a still further embodiment, the present fied polynucleotide comprising any of: (a) the nucleic acid invention provides a modified polynucleotide comprising: (a) sequence of any of SEQID NOS: 1, 3 or 5; (b) an immuno the nucleic acid sequence of any of SEQID NOS: 1.3 or 5; (b) genic encoding portion of (a); or (c) a nucleic acid sequence an immunogenic encoding portion of (a); or (c) a nucleic acid that hybridizes under Stringent conditions to any of (a) or (b). sequence that hybridizes under Stringent conditions to any of 0031. According to yet another embodiment, the present (a) or (b). invention provides a composition comprising a recombi 0025. According to another embodiment, the present nantly expressed protein or polypeptide encoded by a modi invention provides a composition comprising: a modified fied polynucleotide comprising a nucleic acid sequence com polynucleotide comprising a nucleic acid sequence in which prising Surrogate codons in which the nucleotides cytosine the nucleotides cytosine (C) or guanine (G) occupy the (C) or guanine (G) occupy the wobble position in place of the wobble position of Surrogate codons in place of the corre corresponding nucleotides adenine (A), uracil (U) or thymine sponding nucleotides adenine (A), thymine (T) or uracil (U) (T) of a naturally-occurring polynucleotide that expresses the in the nucleic acid sequence of a naturally-occurring poly same protein or polypeptide as said recombinant polynucle nucleotide that expresses the same protein or polypeptide as otide. said recombinant polynucleotide; and a pharmaceutically 0032. According to a further embodiment, the present acceptable buffer, diluent, adjuvant, carrier and/or vector. invention provides a composition comprising an antibody 0026. According to yet another embodiment, the present that immunospecifically binds to a recombinantly expressed invention provides a composition comprising a modified protein of the invention. polynucleotide comprising a nucleic acid sequence in which 0033 According to an even further embodiment, the each codon encoding alanine is GCG, each codon encoding present invention provides a composition prepared by a pro arginine is CGG or AGG, each codon encoding leucine is cess comprising inserting into a vector a modified nucleic CTC, each codon encoding proline is CCT or CCG, each acid sequence comprising Surrogate codons in which the codon encoding glutamic acid is GAG, each codon encoding nucleotides cytosine (C) or guanine (G) occupy the wobble glycine is GGG, each codon encoding isoleucine is ATT, each position in place of the corresponding nucleotides adenine codon encoding serine is TCC, each codon encoding threo (A), uracil (U) or thymine (T) of a naturally-occurring poly nine is ACG, and each codon encoding valine is GTC; and a nucleotide that expresses the same protein or polypeptide as pharmaceutically acceptable buffer, diluent, adjuvant, carrier said modified polynucleotide. and/or vector. 0034. According to a still further embodiment, the present 0027. According to a further embodiment, the present invention provides a composition prepared by a process com invention provides a composition comprising a pharmaceuti prising: inserting into a vector a modified nucleic acid cally acceptable buffer, diluent, adjuvant, carrier and/or vec sequence in which each codon encodingalanine is GCG, each tor; and a modified polynucleotide comprising a nucleic acid codon encoding arginine is CGG or AGG, each codon encod sequence having the general formula: —(X), (Y), (X), ; ing leucine is CTC, each codon encoding proline is CCT or wherein X represents non-surrogate codons having the CCG, each codon encoding glutamic acid is GAG, each nucleic acid sequence of any of the corresponding wild-type codon encoding glycine is GGG, each codon encoding iso codons in the naturally-occurring polynucleotide that encode leucine is ATT, each codon encoding serine is TCC, each the same protein or polypeptide as said modified polynucle codon encoding threonine is ACG, and each codon encoding otide, said wild-type codons having cytosine (C) or guanine valine is GTC. (G) in the wobble position; wherein Y represents surrogate 0035. According to another embodiment, the present codons having a nucleic acid sequence that is different from invention provides a composition prepared by a process com the corresponding wild-type codons in the naturally-occur prising: inserting into a vector a polynucleotide comprising a ring polynucleotide that encode the same protein or polypep modified nucleic acid sequence having the general formula: tide as said modified polynucleotide, said wild-type codons —(X), (Y), (X), ; wherein X represents non-surrogate having adenine (A), uracil (U) or thymine (T) in the wobble codons having the nucleic acid sequence of any of the corre position, said Surrogate codons having cytosine (C) or gua sponding wild-type codons in the naturally-occurring poly nine (G) in the wobble position and encoding the same amino nucleotide that encode the same protein or polypeptide as said US 2009/006925.6 A1 Mar. 12, 2009 modified polynucleotide, said wild-type codons having SEQID NO:14; or (b) a nucleic acid sequence that hybridizes cytosine (C) or guanine (G) in the wobble position; whereinY to SEQID NO:14 under stringent conditions. represents Surrogate codons having a nucleic acid sequence that is different from the corresponding wild-type codons in BRIEF DESCRIPTION OF THE DRAWINGS the naturally-occurring polynucleotide that encode the same protein or polypeptide as said modified polynucleotide, said 0044 FIG. 1 is a graph comparing the expression of pro wild-type codons having adenine (A) or uracil (U) in the tein from the recombinant HIV-1 6106 env gp160 gene pre wobble position, said Surrogate codons having cytosine (C), pared in accordance with an embodiment of the present guanine (G) or thymine (T) in the wobble position and encod invention relative to the expression of protein from the wild ing the same amino acid as the corresponding wild-type type gp160 gene and gp160 gene having modified inhibitory codons in the naturally-occurring polypeptide that encodes Sequences. the same protein or polypeptide as said modified polynucle 0045 FIG. 2 is a plasmid map of the plasmid construct of otide; wherein i is any positive integer of at least 0; and SEQID NO:7. whereinj is any positive integer of at least 1. 0046 FIG. 3 is a plasmid map of the plasmid construct of 0036. According to yet another embodiment, the present SEQID NO:8. invention provides a composition prepared by a process com 0047 FIG. 4 is a plasmid map of the plasmid construct of prising: inserting into a vector any of: (a) the nucleic acid SEQID NO:9. sequence of any of SEQID NOS: 1, 3 or 5; (b) an immuno 0048 FIG. 5 is a plasmid map of the plasmid construct of genic encoding portion of (a); or (c) a nucleic acid sequence SEQID NO:10. that hybridizes under Stringent conditions to any of (a) or (b). 0049 FIG. 6 is a graph comparing expression of protein 0037 According to a further embodiment, the present from IL-15 modified polypeptide (LP) with an IgE leader invention provides for the use of a composition in the prepa sequence in accordance with an embodiment of the present ration of a medicament for inducing an immune response in a invention relative to the expression of protein from alternative mammal. IL-15 constructs in (a) RD cells; (b) COS7 cells, and (c) Hela cells. 0038 According to another embodiment, the present 0050 FIG. 7 is a graph comparing expression of protein invention provides for the use of a composition in the prepa from IL-15 modified polypeptide (LP) with an IgE leader ration of a medicament for treating a condition in a mammal. sequence in accordance with an embodiment of the present 0039. According to a still further embodiment, the present invention relative to the expression of protein from alternative invention provides a transformed, transfected, lipofected or IL-15 constructs in (a) RD cells, and (b) 293 cells. infected cell line comprising: a recombinant cell that 0051 FIG. 8 is a table comparing expression (fold expresses any of: (a) the nucleic acid sequence of any of SEQ increase) of protein from IL-15 modified polypeptide (LP) ID NOS: 1, 3 or 5; (b) an immunogenic encoding portion of with an IgE leader sequence in accordance with an embodi (a); or (c) a nucleic acid sequence that hybridizes under Strin ment of the present invention relative to the expression of gent conditions to any of (a) or (b). protein from alternative IL-15 constructs in RD cells, COS7 0040. According to another embodiment, the present cells, Hela cells, and 293 cells. invention provides a modified polynucleotide comprising: (a) 0.052 FIG. 9 is a graph comparing expression of protein the nucleic acid sequence of any of SEQID NOS: 12-16; (b) from IL-15 modified polypeptide (LP) with an IgE leader an immunogenic encoding portion of (a); or (c) a nucleic acid sequence in accordance with an embodiment of the present sequence that hybridizes under Stringent conditions to any of invention relative to the expression of protein from alternative (a) or (b). IL-15 constructs in a CTLL2 mouse cell proliferation assay. 0041 According to yet another embodiment, the present 0053 FIG. 10 is a graph comparing in vivo expression of invention provides a composition that comprises a modified protein from IL-15 modified polypeptide (LP) with an IgE polynucleotide comprising: (a) a non-native leader sequence; leader sequence in accordance with an embodiment of the and (b) a nucleic acid sequence comprising cytosine (C) or present invention relative to the expression of protein from guanine (G) at the wobble position of at least one codon that alternative IL-15 over time. encodes any of the amino acids alanine, arginine, leucine, proline, glutamic acid, glycine, isoleucine, serine, threonine, 0054 FIG. 11 is a plasmid map for the O-IL-15-IgE leader or valine where adenine (A), uracil (U) or thymine (T) occupy plasmid construct according to an embodiment of the present the wobble position of the corresponding codon of the natu invention. rally-occurring nucleic acid sequence. 0055 FIG. 12 is a plasmid map for the LP-IL-15-IgE 0042. According to a further embodiment, the present leader plasmid construct according to an embodiment of the invention provides a composition that comprises a recombi present invention. nant polynucleotide comprising: (a) an IgE leader sequence; 0056 FIG. 13 is a plasmid map for the BH-15-IgE leader and (b) a nucleic acid sequence comprising cytosine (C) or plasmid construct according to an embodiment of the present guanine (G) at the wobble position of at least one codon that invention. encodes any of the amino acids alanine, arginine, leucine, proline, glutamic acid, glycine, isoleucine, serine, threonine, DETAILED DESCRIPTION OF THE INVENTION or valine where adenine (A), uracil (U) or thymine (T) occupy 0057. An appropriate level of a protein in mammalian cells the wobble position of the corresponding codon of the natu is essential in vivo for enhanced immunological and/or thera rally-occurring nucleic acid sequence. peutic responses, e.g., the use of the gene and its protein 0043. According to a still further embodiment, the present product as an immunogen, DNA vaccine, co-immunogen, invention provides a composition comprising: a polynucle adjuvant, carrier protein or vector, therapeutic agent, diagnos otide comprising (a) a nucleic acid sequence having at least tic agent, therapeutic, immuno-prophylactic, immuno-thera about 70% sequence identity to the nucleic acid sequence of peutic, etc., as well as for in vitro recombinant protein expres US 2009/006925.6 A1 Mar. 12, 2009 sion purposes, e.g., the use of the gene and its protein product that expresses Substantially the same protein or polypeptide in assays, tests, diagnostics, research tools, etc. The efficiency as said modified polynucleotide (or a functionally equivalent of a gene in expressing its protein product is a controlling protein or polypeptide, as would be known to a person of skill factor in the attainment of appropriate levels of the protein in in the art). The modified polynucleotide of the invention need cells. Certain wild-type genes fail to provide appropriate pro not be an exact replica of the wild-type ORF wherein every tein levels in mammalian cells. The present invention is codon having A or U in the wobble position is substituted with directed to improving the expression efficiency of such genes. a Surrogate codon. Merely a sufficient number of Surrogate 0058 An effective IL-15 plasmid for DNA vaccination codons in place of naturally occurring codons to achieve that secretes enhanced levels of IL-15 was unexpectedly iden enhanced gene expression is necessary. tified. In particular, it was found that 1) the replacement of 0063 A minimally sufficient number of surrogate codons native signal peptide with the Human IgE leader sequence; 2) or any number greater than that amount is contemplated by non preferred codons are replaced with either optimized or the invention. A Suitable number of Surrogate codons for a less preferred codons while preserving the native amino acid polynucleotide in accordance with the present invention is sequence; 3) the nucleotide sequence was modified to reduce readily determined by one of skill by routine testing. It is not the secondary mRNA structure for improved translation. necessary that a predetermination of a specific number of Surrogate codons be made. However, a predetermined num Modified Polynucleotides ber of replacements may be used in the interest of efficiency. 0059. As described herein, the inventors have devised For example, in constructing a polynucleotide of the inven modified polynucleotides that provide unexpectedly tion, one may predetermine that a specified percentage of the improved gene expression in mammalian cells both in vitro codons of the ORF may be re-engineered, for example, about and in vivo for various poorly-expressed genes. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 0060. These polynucleotides represent a new version of a 96%, 97%, 98%, 99% or 100% of the codons, without limi wild-type gene. In particular, the inventors discovered that tation, may be the Subject of re-engineering. Normally, at enhanced expression was unexpectedly provided by a new least 10% of the codons are the Subject of reengineering (e.g., version of a gene in the form of a synthesized polynucleotide 10% of the ORF is the new version of the gene while the which comprises “surrogate codons' in the open reading remaining 90% is the same as or functionally the same as the frame (ORF) of the gene sequence, wherein the “surrogate wild-type ORF). In certain embodiments, at least about 50% codons' still encode identical amino acid residues (although of the codons are the Subject of re-engineering. In other biologically equivalent amino acid sequences/proteins, Sub embodiments, at least about 90% of the codons are the subject stantially identical amino acid sequences/proteins, etc. are of re-engineering with Surrogate codons. also contemplated by the present invention, as described in 0064. The surrogate codons of the present invention are further detail below). the non-naturally-occurring codons (of a gene) that encode 0061. A “surrogate codon’, as used herein, refers to a for the following amino acids: alanine (Ala), asparagine or codon for an ORF, other than the naturally occurring (i.e., aspartate (ASX), cysteine (CyS), aspartate (Asp), glutamate wild-type) codon when that wild-type codon has an A, T (in (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), the case of DNA) or U (in the case of RNA) in the wobble isoleucine (lie), lysine (LyS), leucine (Leu), methionine position, but encoding the same amino acid as that corre (Met), asparagine (ASn), proline (Pro), glutamine (Gln), argi sponding naturally occurring codon (i.e., the codon at the nine (Arg), serine (Ser), threonine (Thr), tyrosine (Tyr), or same position in the wild-type ORF). As used herein, the glutamine or glutamate (Glx). In a particular embodiment, the terms, “naturally-occurring and “wild-type' are used inter Surrogate codons of the invention are the non-naturally-oc changeably herein. In certain embodiments, the Surrogate curring codons (of a gene) with C or G in the wobble position codon has C or G in its wobble position. In another embodi that encode for any of alanine (Ala), asparagine or aspartate ment, the Surrogate codon is not a "preferred codon’ as (ASX), cysteine (CyS), aspartate (Asp), glutamate (Glu), phe defined by Seed et al. The surrogate codons of the present nylalanine (Phe), glycine (Gly), histidine (His), lysine (LyS), invention are used in modified polynucleotides in place of leucine (Leu), methionine (Met), asparagine (ASn), proline corresponding disfavored codons, e.g., the naturally-occur (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine ring codon with A or T (if DNA) or U (if RNA) in the wobble (Thr), tyrosine (Tyr), or glutamine or glutamate (Glx), with position, of the wild-type form of the gene, for certain of the out limitation. A recombinant polynucleotide of the invention amino acids as described below. As used herein, the “wobble' need not include Surrogate codons for each amino acid position of a codon is the third nucleotide position of a codon encoded. Select Surrogate codons that encode any number of triplet, as read in the 5' to 3’ direction. amino acids may be predetermined for inclusion in the 0062. The invention disclosed herein utilizes a general recombinant version of the gene provided that the objective of approach directed to modified forms of a gene (i.e., recom improving expression of the gene is achieved. A person of binant polynucleotides). According to this general approach, skill in the art would be able to determine through routine modified polynucleotides are formed. These polynucleotides testing a minimally effective number. In one particular comprise a nucleic acid sequence comprising Surrogate embodiment, each of the codons for alanine (Ala), asparagine codons in place of at least Some of the codons of the corre or aspartate (ASX), cysteine (CyS), aspartate (Asp), glutamate sponding wild-type polynucleotide for the gene. For (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), example, in accordance with embodiments of the invention, a isoleucine (lie), lysine (LyS), leucine (Leu), methionine modified polynucleotide comprises a nucleic acid sequence (Met), asparagine (ASn), proline (Pro), glutamine (Gln), argi comprising Surrogate codons in which the nucleotides nine (Arg), serine (Ser), threonine (Thr), tyrosine (Tyr), or cytosine (C) or guanine (G) occupy the wobble position in glutamine or glutamate (Glx) is replaced with a Surrogate place of the corresponding nucleotides adenine (A) or uracil codon to form the recombinant version of the gene in accor (U) or thymine (T) of a naturally-occurring polynucleotide dance with an embodiment of the invention. US 2009/006925.6 A1 Mar. 12, 2009

0065 Accordingly, in the present invention, it is unneces Tymovirus, Umbravirus, Viroids, , Tailed sary to replace each codon that has A, T or U in the wobble Phages, and as yet unclassified , without limitation. position for every amino acid, Substitute in specifically deter 0070. In one embodiment of the invention, a viral gene is mined “preferred codons' or remove inhibitory sequences. associated with lentiviruses, , herpes viruses, 0066. In certain embodiments, the surrogate codons used adenoviruses, adeno-associated viruses, vaccinia , or in the modified polynucleotides of the present invention are baculovirus, without limitation. In certain embodiments, viral genes include, for example, those of Human immuno those that encode alanine, arginine, leucine, proline, glutamic deficiency virus, Simian immunodeficiency virus, Respira acid, glycine, isoleucine, serine, threonine and valine. In tory syncytial virus, Parainfluenza virus types 1-3, Influenza other embodiments, the Surrogate codons used in the poly virus, Herpes simplex virus, Human cytomegalovirus, Hepa nucleotides of the invention are those that encode alanine, titis A virus, Hepatitis B virus, Hepatitis C virus, Human arginine, leucine, proline, glycine, isoleucine, serine, threo papillomavirus, poliovirus, rotavirus, caliciviruses, Measles nine and valine. In one particular embodiment, the Surrogate virus, Mumps virus, Rubella virus, adenovirus, rabies virus, codons used in the modified polynucleotides of the invention vesicular stomatitis virus, canine distemper virus, rinderpest are those that encode alanine, arginine, leucine, proline, gly virus, Human metapneumovirus, avian pneumovirus (for cine, serine, threonine and valine. merly turkey rhinotracheitis virus), Hendra virus, Nipah 0067. In accordance with an embodiment of the invention, virus, coronavirus, parvovirus, infectious rhinotracheitis the Surrogate codons are a randomized selection of at least viruses, feline leukemia virus, feline infectious peritonitis about 10% of the codons in said modified polynucleotide that virus, avian infectious bursal disease virus, Newcastle disease encode for any of the amino acids alanine, arginine, leucine, virus, Marek's disease virus, porcine respiratory and repro proline, glycine, isoleucine, serine, threonine and valine. In ductive syndrome virus, equine arteritis virus and various accordance with another embodiment, the Surrogate codons Encephalitis viruses, without limitation. area randomized selection of at least about 50% of the codons 0071 Specific viral genes contemplated by the present in said polynucleotide that encode for any of the amino acids invention include, for example, any of the genes of HIV or any alanine, arginine, leucine, proline, glycine, isoleucine, serine, of the genotypes of HPV, including high-risk and low-risk threonine and valine. In a further embodiment, the Surrogate genotypes. For example, genes of HIV contemplated by the codons area randomized selection of at least about 90% of the invention include gag, pol, env, tat, rev, vif, nef, vpr, vpu and codons in said polynucleotide that encode for any of the Vpx, without limitation. Genes of HPV contemplated by the amino acids alanine, arginine, leucine, proline, glycine, iso invention include, for example, E1, E2, L1, L2, E6 and E7 leucine, serine, threonine and valine. In yet another embodi without limitation. The genotypes of HPV contemplated by ment, the Surrogate codons are each of the codons in said the present invention include, for example, high-risk geno polynucleotide (i.e., 100%) that encode for the amino acids types, such as HPV 16, 18, 31, 33, 45, 52, 56 or 58 and alanine, arginine, leucine, proline, glycine, isoleucine, serine, low-risk genotypes, such as 6 and 11, without limitation. threonine and valine. According to an embodiment, the gene is the human papillo 0068. The present invention contemplates embodiments mavirus 16 (HPV16) E7 gene (E7), or human immuno-defi directed to any gene that is poorly expressed or any gene for ciency virus (HIV-1) gag gene (gag) or gp160 envelope gene which improved levels of protein expression is desirable for (env). Compositions, fusion constructs or any other multi in vivo and/or in vitro uses. For example, a Subject gene may gene structures containing any combination of the foregoing be a viral, bacterial, protist, fungal, plant or animal gene, are also contemplated by the present invention. without limitation. Any Such gene that is poorly expressed in 0072 Specific bacterial genes include the genes of any mammalian cells is contemplated by the present invention. bacterial species, including for example, without limitation, 0069. In the case of viral genes, without limitation, the Haemophilus influenzae (both typable and nontypable), Hae viral gene may be associated with a DNA (double stranded or mophilus somnus, Moraxella catarrhalis, Streptococcus single stranded) or RNA (double stranded or single stranded) pneumoniae, Streptococcus pyogenes, Streptococcus agalac virus, without limitation. Viral genes of viruses from any viral tiae, Streptococcus faecalis, Helicobacter pylori, Neisseria family are contemplated by the present invention, including, meningitidis, Neisseria gonorrhoeae, Chlamydia trachoma for example, , Arenaviridae, Arterivirus, Astro tis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella viridae, , Badnavirus, , Brinaviri pertussis, Alloiococcus Otiditis, Salmonella typhi, Salmonella dae, , Bunyaviridae, , Capillovi typhimurium, Salmonella choleraesuis, Escherichia coli, Shi rus, Carlavirus, Caulimovirus, , , gella, Vibrio cholerae, Corynebacterium diphtheriae, Myco Comoviridae, , Corticoviridae, Cystoviridae, bacterium tuberculosis, Mycobacterium avium-Mycobacte Deltavirus, Dianthovirus, Enamovirus, , Flaviviri rium intracellulare complex, Proteus mirabilis, Proteus dae, , , , Hepadnaviri vulgaris, Staphylococcus aureus, Staphylococcus epidermi dae, , Hordeivirus, Hypoviridae, Idaeovirus, dis, Clostridium tetani, Leptospira interrogans, Borrelia Inoviridae, , Leviviridae, , burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Luteovirus, Machlomovirus, Marafivirus, , Actinobacillus pleuropneumoniae and Mycoplasma gallisep , Necrovirus, , , ticum. Papovaviridae, , , Parvaviri 0073. Further, the present invention is applicable to any dae, , Picornaviridae, , gene which is a suitable subject for improved efficiency in the , Polydnaviridae, , , Pox manner of the present invention, i.e., engineering a recombi yiridae, , Retroviridae, , Rhizidio nant polynucleotide for the gene with Surrogate codons in virus, Sequiviridae, , Sobemovirus, Tectiviridae, place of naturally occurring codons with A or U in the wobble Tenuivirus, Tetraviridae, Tobamovirus, Tobravirus, position. Thus, although the term “poorly-expressed’ genes Togavridae, , , Trichovirus, is used throughout, the present invention is by no means US 2009/006925.6 A1 Mar. 12, 2009

intended to be limited to genes that meet some threshold location of inhibitory sequences and there are no straightfor requirement of poor expression. Instead, modified polynucle ward prescriptions from the gag work to predict how to elimi otides directed to poorly-expressed genes are merely exem nate inhibitory sequences from these genes. plary to illustrate the dramatic improvement in protein levels 0077 According to an embodiment of the present inven in the circumstances where such improvement is most perti tion, codons throughout a gene sequence are replaced (e.g., nent. Therefore, the present invention contemplates applica Surrogate codons replace wild-type codons in a modified bility to genes that may not be considered to be poorly construct) without the need to identify and then mutate inhibi expressed by persons skilled in the art, as well as to those that tory sequences (as performed for gag) and without altering are generally considered or proven to be poorly-expressed, every codon by use of preferred codons (as performed for without limitation. env). When a naturally occurring disfavored codon (e.g., with 0074. Upon selection of a desired target gene of a desired A or U in the wobble position) is replaced with (i.e., its species (e.g., the E1 gene of HPV 16), a person of skill in the position in the modified form is occupied by) a “surrogate art, based upon the guidance provided herein, would be able codon’ encoding the same amino said, there is an opportunity to formulate the sequence of a desired recombinant in accor to eradicate inhibitory sequence(s), instability sequence(s), dance with an embodiment of the present invention. The and/or provide codons that are more efficiently translated sequencing is performed for example, by handloris computer than their naturally occurring counterparts. assisted. A person of skill in the art may make a replacement 0078. It was surprisingly discovered that alteration of all at each disfavored wobble position, or at Some percentage of possible codons and utilization of “preferred’ codons was not the disfavored wobble positions. For example, the first 50% of necessary to achieve improved protein levels expressed by the disfavored wobble positions or the second 50% of disfavored genes cited above. Thus, it is possible to exploit the degen wobble positions. The modified sequence is tested by routine eracy of the genetic code to develop recombinant polynucle methods to determine whether the percentage change pro otides with improved protein expression of a gene relative to vides a desired level of expression. The examples herein the wild-type polynucleotide of the gene (or other recombi provide guidance as to Such testing, however, it is well within nant polynucleotides for the gene). Thus, it is unnecessary to the abilities of a person of skill in the art to conduct such construct a complete “codon optimized' version of gp120 routine testing in a variety of ways. In certain embodiments, envelope as previously described (see Haas et al., Andre etal.) replacement is made at each disfavored wobble position, thus in which non-preferred wild-type codons from env were eliminating the need to select certain portions of the gene and replaced with “preferred codons to enhance protein levels certain percentages of wobble positions for replacement. expressed by the gene. Once the sequence of the polynucleotide is determined, it is 0079 Table I below lists non-limiting examples of surro well within the ability of a person of skill in the art to prepare gate codons of the present invention. In particular, Table I the modified polynucleotide using well known techniques shows the Surrogate codons for ten of the twenty L-amino and methods, as further described in the examples below. acids that have been utilized as replacements for existing 0075 Several poorly-expressed viral genes illustrate the disfavored codons, according to an implementation of the benefits of the present invention. For example, the following present invention. In accordance with this embodiment of the wild-type viral genes demonstrate poor expression in mam invention, codons encoding the remaining ten amino acids malian cells: human papillomavirus 16 (HPV 16) E7, human were not replaced by surrogate codons in the modified form of immuno-deficiency virus type-1 (HIV-1) gag and gp160 (en the gene. velope) (hereafter denoted E7, gag, and env, respectively). In each of these wild-type genes, the naturally occurring nucleic TABLE I acid sequences of the genes are AU rich and biased toward “disfavored codons’ (containing an A or U in the 3d or SURROGATE CODONS “wobble' position of the codon nucleotide triplet). As noted Amino acid Amino acid above, mammalian genes that express proteins at high levels Codon encoded Codon encoded have a G/C preference in the wobble position. Thus, these GCG Alanine GAG Glutamic Acid wild-type genes with A or U in the wobble position may not CGG or AGG Arginine GGG Glycine be handled efficiently by the mammalian translational CTC Leucine ATT Isoleucine machinery. CCT or CCG Proline TCC Serine 0076 Further, as discussed above, separately from the ACG Threonine GTC Valine translational mechanisms accounting for poorly-expressed genes, there have been various AU rich RNA instability 0080. In accordance with an embodiment of the present sequences discovered in several messenger RNAs (mRNAs) invention, recombinant polynucleotides were prepared in which do not directly impact the translatability of a given which disfavored codons (A or Uat the wobble position) were mRNA but limit protein expression by increasing mRNA replaced by the surrogate codons listed in Table I above for turnover. In addition, several specific “inhibitory” sequences the amino acid encoded by the disfavored codon, and the contained within the HIV-1 gag ORF have been described corresponding new (i.e. modified) nucleic acid sequence was (see Pavlakis) which limit the expression levels of gag by created by joining oligonucleotides encoding the new inhibiting nuclear export of these transcripts. Codons encom sequence and assembling the fragments to create the modified passing these inhibitory sequences are difficult to selectively polynucleotide comprising the new sequence. replace to eliminate the inhibitory effect because the I0081. The recombinant ORF was cloned into a plasmid sequence motifs that define either instability or inhibitory DNA expression vector that allowed in vitro expression-stud sequences are not easily identified. Moreover, several genes ies for comparing the levels of protein expression of the (e.g. E7 and En among others) which appear to also contain modified polynucleotide and the wild-type polynucleotide. inhibitory sequences have not yet been mapped to identify the Transient transfection assays (data not shown) performed US 2009/006925.6 A1 Mar. 12, 2009 with several cell lines revealed increases in protein expression recombinant nucleic acid sequence. The fragments are levels for three gene products (i.e., E7, gag, and env) when hybridized and subsequently filled in by a DNA polymerase their gene sequence was modified as described above. The (such as Pfix Turbo, Invitrogen). This staggered, overlapping increased protein expression (as measured by Western blot, arrangement of the fragments is then ligated, for example, ELISA and the like) demonstrated by the altered codon con using a heat stable ligase (Ampligase). structs compared to wild-type (naturally occurring) construct I0086 Specific protocols for preparing the polynucleotides for three different genes indicated that this method is appli of the present invention are provided in the Examples below. cable to a variety of poorly expressed proteins. These specific protocols are merely illustrative. A person 0082 In recognition that several codon choices are pos skilled in the art would readily be able to employ a variety of sible for some of the twenty amino acids, for example, the suitable techniques to accomplish the objectives of the amino acids alanine, arginine, glycine, glutamic acid, isoleu present invention, upon grasping the inventive concepts dis cine, leucine, proline, serine, threonine, and valine, an closed herein. All Such suitable techniques for preparing embodiment of the present invention is directed to the codons recombinant polynucleotides are contemplated by the present encoding those amino acids. Thus, in accordance with an invention. embodiment of the invention, a modified polynucleotide has I0087. According to an embodiment of the invention, the a nucleic acid sequence, which differs from that of the wild leader sequence of the polynucleotide is altered or substituted type sequence, in which each codon, that corresponds to a with a non-native leaders sequence. For example, a non naturally-occurring codon having A, U or T in the wobble native leader sequence is added to a modified polynucleotide position, encoding alanine is GCG, each codon encoding of the presents invention and replaces the native leader arginine is CGG or AGG, each codon encoding leucine is sequence of the polynucleotide. Thus, the present invention CTC, each codon encoding proline is CCT or CCG, each contemplates a modified polynucleotide comprising a non codon encoding glutamic acid is GAG, each codon encoding native leader sequence. The non-native leader sequence may glycine is GGG, each codon encoding isoleucine is ATT, each be any Suitable sequence or combination thereofthat provides codon encoding serine is TCC, each codon encoding threo enhanced expression. It has been Suprisingly found that the nine is ACG, and each codon encoding valine is GTC. combination of modifying the polynucleotides using Surro 0083. In certain other embodiments, codons for amino gate codons as described herein with the use of a non-native acids other than the ten listed above also serve as Surrogate leader sequence provides Synergistically improved expres codons. In other words, replacement of the naturally-occur sion, as described in Example 5 below. The non-native leader ring codons, with A, U or T in the wobble position, encoding sequence may be human non-native leader sequence. The other amino acids is contemplated. It is also contemplated non-native leader sequence may be an immunoglobulin that certain embodiments of the invention provide Surrogate leader sequence. codons for only some of the ten amino acids listed in Table I. I0088 According to an embodiment, the non-native leader Upon grasping the concept of the invention as fully described sequence is (a) an IgE leader sequence or (b) a leader herein, a person skilled in the art would routinely be able to sequence that hybridizes to an IgE leader sequence under determine a minimally or optimally desired number of stringent conditions. According to another embodiment, the codons through routine methods, based upon the guidance non-native leader sequence is: (a) a leader sequence having provided herein. In certain embodiments, the polynucleotides SEQ ID NO:11; or (b) a leader sequence that hybridizes to of the present invention comprise Surrogate codons for just SEQ ID NO:11 under stringent conditions. The non-native the nine amino acids, alanine, arginine, glycine, isoleucine, leader sequence has at least 70%, 80%, 90%, 95%,97%.98% leucine, proline, serine, threonine, and valine in place of each or 99% sequence identity to the nucleic acid sequence of SEQ of the corresponding codons having A or U in the wobble ID NO:11 according to other embodiments of the present position. It should be noted, however, that any changes to invention. According to another embodiment, the non-native those changed codons and/or the other codons that permit the leader sequence has the nucleic acid sequence of SEQ ID protein to retain its functionality are contemplated by the NO:11. A person skilled in the art would readily be able to present invention. Examples of Such changes are provided construct or alter a polynucleotide to include a non-native below. leader sequence in the manner of the present invention, based 0084. The modified polynucleotides of the invention are upon the guidance provided herein. prepared in any Suitable manner as would be known to per I0089. The polynucleotides are prepared in various forms Sons skilled in the art. For example, the present invention (e.g., single-stranded, double-stranded, vectors, probes, contemplates the use of chemical synthesis, nucleotide Sub primers) as desired. The term “polynucleotide' includes any stitution, codon Substitution, DNA libraries, mutagenesis, strand of DNA and RNA, single stranded and double isolation and purification from native entity, etc. and any Stranded, and also their analogs, such as those containing combinations thereof, without limitation. modified backbones. The term “modified polynucleotide’ as 0085. In one embodiment, a full length polynucleotide used herein, describes any strand of DNA or RNA, including sequence is determined by selecting Surrogate codons for the single or double stranded, that are recombinantly prepared or disfavored codons. This may be done by hand, computer that have been altered from their naturally-occurring state assisted or any other method. Once the desired sequence is (through insertion, deletion, Substitution, etc.) with Surrogate determined, then oligonucleotides comprising fragments of codons or as otherwise consistent with the embodiments of the determined sequence are obtained or prepared. Such oli the present invention as described herein. The DNA may be of gonucleotides are readily obtained from commercial vendors, any type, such as cDNA, genomic DNA, synthesized DNA, such as InvitrogenTM (Carlsbad, Calif.). The fragments are isolated DNA or a hybrid thereof. The RNA may be also be of selected Such that they can form a staggered, overlapping any type RNA molecule such as mRNA. The constructs of the arrangement. The modified polynucleotides are synthesized present invention contemplate any regulator elements neces by joining oligonucleotides that comprise fragments of the sary or desirable for expression of the sequence, such as a US 2009/006925.6 A1 Mar. 12, 2009 promoter, an initiation codon, a stop codon, and a polyade 0093. For example, according to an embodiment of the nylation signal, for example, without limitation. Any Suitable present invention, the modified polynucleotides comprise a enhancer is also contemplated by the present invention. Non nucleic acid sequence that is identical to any of the reference limiting exemplary enhancers include human Actin, human sequences of odd numbered SEQID NOS: 1-5 or any of SEQ Myosin, human Hemolobin, human muscle creatine, and ID NOS:12-16 (which are sequences modified in accordance viral enhancers such as those from CMV, RSV and EBV. with the invention), that is 100% identical, or it may include 0090. Several specific recombinant polynucleotides, a number of nucleotide alterations (e.g. at least 99%, 98%, including specific nucleic acid sequences, for various viral 97%, 96%. 95%, 94%, 90%, 85%, 80%, 70%, or 60% iden genes are provided herein. These are merely exemplary and tical, etc.) as compared to the reference sequence. Such alter the invention is not intended to be limited thereto. Rather, the ations are selected from the group consisting of at least one inventive concept is broadly applicable as described herein. nucleotide deletion, Substitution, including transition and Moreover, the present invention contemplates modified poly transversion, or insertion, and wherein said alterations occur nucleotides which are variations on any of the recombinant at the 5' or 3' terminal positions of the reference nucleotide polynucleotides described herein, such as, for example, the sequence or anywhere between those terminal positions, specifically disclosed sequences, without limitation. For interspersed either individually among the nucleotides in the example, these would include variations wherein the variant reference sequence or in one or more contiguous groups nucleic sequence encodes a different amino acid sequence within the reference sequence. The number of nucleotide than the specifically disclosed sequence, however, the func alterations is determined by multiplying the total number of tionality of the different amino acid sequence is the same as nucleotides in any of odd numbered SEQID NOS:1-5 or any that encoded by the sequence described herein. of SEQID NOS:12-16 by the numerical percent of the respec 0091. According to an embodiment the modified poly tive percent identity (divided by 100) and subtracting that nucleotide expresses a viral polypeptide. The present inven product from said total number of nucleotides in said tion contemplates modified polynucleotides from any agent Sequence. or organism, Such as pathogenic organisms, for example, 0094 Certain embodiments of the invention relate to poly HIV, HSV. HCV, WNV or HBV. For example, according to an nucleotides and sequence modifications thereof. In one embodiment immunogenic compositions are prepared from embodiment, a polynucleotide of the invention is a polynucle the pathogenic organisms for the purpose of immunizing an otide comprising a nucleotide sequence having functional individual against the pathogen. For example, the modified equivalency and at least about 95% identity to a nucleotide polynucleotide may express the viral polypeptides HPV 16 sequence chosen from one of the odd numbered SEQ ID HIV-1 or gp160 or any combinations thereof, without limita NO:1-5 or any of SEQID NOS:12-16, a degenerate variant tion. According to an embodiment, a modified polynucleotide thereof, or a fragment thereof. As defined herein, a “degen may comprise the ORF for HPV 16 E7 gene. According to erate variant' is defined as a polynucleotide that differs from another embodiment, a modified polynucleotide comprises the nucleotide sequence shown in the odd numbered SEQID the ORF for the HIV-1 gag gene. According to another NOS:1-5 or any of SEQ ID NOS:12-16 (and fragments embodiment, a modified polynucleotide comprises the ORF thereof) due to degeneracy of the genetic code, but still for the gp160 envelope gene. encodes the same protein (e.g., the even numbered SEQ ID 0092. According to an embodiment, the modified poly NOS: 2-6) as that encoded by the nucleotide sequence shown nucleotide encodes for a cytokine, growth factor, lymphok in the odd numbered SEQ ID NOS: 1-5 or any of SEQ ID ine, Such as alpha-interferon, gamma-interferon, GM-CSF, NOS:12-16. platelet derived growth factor, TNF, EGF, ILA, IL-2, IL-4, 0095. In other embodiments, the polynucleotide is a IL-6, IL-10, IL-12, IL-15 as well as fibroblast growth factor, complement to a nucleotide sequence chosen from one of the Surface active agents such as immune-stimulating complexes odd numbered SEQID NOS: 1-5 or any of SEQID NOS:12 (ISCOMS), Freunds incomplete adjuvant, LPS analog 16, a degenerate variant thereof, or a fragment thereof. In yet including monophsphoryl Lipid A (WL), muramyl peptides, other embodiments, the polynucleotide is selected from the quinone analogs and vesicles such as squalene and squalene, group consisting of DNA, chromosomal DNA, cDNA and and hyaluronic acid. Any cytokine is contemplated by the RNA and may further comprises heterologous nucleotides. In present invention. According to another embodiment, the another embodiment, an isolated polynucleotide hybridizes cytokine is an interleukin. According to another embodiment, to a nucleotide sequence chosen from one of odd numbered polynucleotide encodes for IL-15 or a peptide or polypeptide SEQ ID NOS: 1-5 or any of SEQID NOS:12-16, a comple having the activity of IL-15. According to another embodi ment thereof, a degenerate variant thereof, or a fragment ment, the modified polynucleotide encodes for IL-15. thereof, under high Stringency hybridization conditions. In According to another embodiment, the modified polynucle yet other embodiments, the polynucleotide hybridizes under otide comprises the nucleic acid sequence of any of SEQID intermediate Stringency hybridization conditions. NOS:12-16. According to another embodiment, the modified 0096. It will be appreciated that polynucleotides of the polynucleotide comprises the nucleic acid sequence of SEQ present invention are obtained from natural Sources (and then ID NO:14. The nucleotide and amino acid sequences of IL-15 altered) or are synthetic or semi-synthetic or some combina are well known and set forth in Campbell, et al. (1987) Proc. tion thereof. Furthermore, the nucleotide sequence is related Natl. Acad. Sci. USA 84:6629-6633, Tanabe, et al. (1987).J. by mutation, including single or multiple base Substitutions, Biol. Chem. 262:16580-16584, Campbell, et al. (1988) Eur.J. deletions, insertions and inversions, to a naturally occurring Biochem. 174:345-352, AZuma, et al. (1986) Nucl. Acids sequence, provided always that the nucleic acid molecule Res. 14:9149-9158, Yokota, et al. (1986) Proc. Natl. Acad. comprising Such a sequence is capable of being expressed as Sci. USA 84:7388-7392, and accession code Swissprot a functionally equivalent polypeptide as described above. A PO5113, which are each incorporated herein by reference in nucleic acid molecule of the invention is RNA, DNA, single their entirety. stranded or double stranded, linear or covalently closed cir US 2009/006925.6 A1 Mar. 12, 2009

cular form. In certain embodiments, the nucleotide sequence known sequence are hybridized, the hybrid length can be has expression control sequences positioned adjacent to it, determined by aligning the sequences of the polynucleotides Such control sequences usually being derived from a heter and identifying the region or regions of optimal sequence ologous source. In other embodiments, the recombinant complementarities. expression of a nucleic acid sequence of the invention include 0099 bufferH: SSPE (1xSSPE is 0.15M NaCl, 10 mM a stop codon sequence, such as TAA, at the end of the nucleic NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted acid sequence. for SSC (1xSSC is 0.15MNaCl and 15 mM sodium citrate) in 0097. According to an embodiment, the invention also the hybridization and wash buffers; washes are performed for includes polynucleotides capable of hybridizing under 15 minutes after hybridization is complete. reduced stringency conditions. According to another embodi 0100 TB through TR: The hybridization temperature for ment the invention includes polynucleotides capable of hybrids anticipated to be less than 50 base pairs in length hybridizing under Stringent conditions, and under another should be about 5-10 C less than the melting temperature embodiment the present invention includes polynucleotides (Tm) of the hybrid, where Tm is determined according to the capable of hybridizing under highly stringent conditions, to following equations. For hybrids less than 18 base pairs in the polynucleotides described above. Examples of stringency length, Tm(C)=2(ii of A+T bases)+4(ii of G+C bases). For conditions are shown in the Stringency Conditions Table hybrids between 18 and 49 base pairs in length, Tm(C)=81. below: highly stringent conditions are those that are at least as 5+16.6(log 10Na+I)+0.41 (% G+C)-(600/N), where N is the stringent as, for example, conditions A-F. Stringent condi number of bases in the hybrid, and Na+ is the concentration tions are at least as stringent as, for example, conditions G-L; of sodium ions in the hybridization buffer (Na+ for and reduced stringency conditions are at least as stringent as, 1xSSC=0.165 M). for example, conditions M-R. 0101 Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Labo TABLE II ratory Manual, Cold Spring Harbor Laboratory Press, Cold HYBRIDIZATION STRINGENCY CONDITIONS Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols Poly- Hybrid Hybridization Wash in Molecular Biology, 1995, F. M. Ausubel et al., eds. John Stringency nucleotide Length Temperature and Temperature Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated Condition Hybrid (bp)I BufferH and BufferH herein by reference. 0102. In certain embodiments, modifications and changes A. DNA:DNA >50 65 C.: 1XSSC-or- 65 C.: 0.3xSSC 42 C.; 1xSSC, 50% are made in the structure of a polynucleotide of the present ormamide invention while retaining functional equivalency (Such as B DNA:DNA <50 TB; 1xSSC TB; 1xSSC immunogenicity, therapeutic benefit, binding affinity, etc) of C DNA:RNA >50 67 C.: 1XSSC-or- 67 C.: 0.3xSSC the protein product encoded by the modified polypeptide. 45 C.; 1xSSC, 50% ormamide Such modifications and changes are fully contemplated by D DNA:RNA <50 TD: 1XSSC TD; 1xSSC the present invention. For example, without limitation, cer E RNA:RNA >50 70 C.: 1XSSC-or- 70 C.: 0.3xSSC tain amino acids can be substituted for other amino acids, 50 C.; 1xSSC, 50% ormamide including nonconserved and conserved Substitution, in an F RNA:RNA <50 TF; 1xSSC Tf 1xSSC amino sequence without appreciable loss of functionality/ G DNA:DNA >50 65 C.: 4xSSC-or- 65 C.; 1xSSC utility (e.g., immunogenicity, therapeutic benefit, etc.) and 42 C.: 4xSSC, 50% thus in the polynucleotide the corresponding codon encoding ormamide those amino acids can be changed accordingly, as would be H DNA:DNA <50 TH; 4xSSC TH: 4xSSC I DNA:RNA >50 67 C.: 4xSSC-or- 67 C.; 1xSSC understood by a person skilled in the art. 45 C.; 4xSSC, 50% 0103) In fact, as it is the interactive capacity and nature of ormamide a polypeptide that defines that polypeptide's biological func J DNA:RNA <50 TJ; 4Xssc TJ; 4xSSC K RNA:RNA >50 70 C.: 4xSSC-or 67 C.; 1xSSC tional activity, a number of amino acid sequence Substitutions 50 C.; 4xSSC, 50% are made in a polypeptide sequence, and thus its underlying ormamide nucleic acid coding sequence, and nevertheless obtain a L RNA:RNA <50 TL: 2Xssc TL:2xSSC polypeptide with like properties. The present invention con M DNA:DNA >50 50 C.: 4xSSC-or- 50 C.; 2xSSC 40 C.; 6xSSC, 50% templates any changes to the structure of the nucleic acid ormamide sequences encoding the Subject polypeptides or proteins, N DNA:DNA <50 TN; 6xSSC TN; 6xSSC wherein the polypeptide or protein retains its functionality or O DNA:RNA >50 55 C.: 4xSSC-or- 55 C.; 2xSSC a biologically equivalent functionality. A person of ordinary 42 C.; 6xSSC, 50% ormamide skill in the art would be readily able to routinely modify the P DNA:RNA <50 TP; 6xSSC TP; 6xSSC disclosed polypeptides and polynucleotides accordingly, Q RNA:RNA >50 60 C.: 4xSSC-or- 60 C.; 2xSSC based upon the guidance provided herein, while remaining 45 C.; 6xSSC, 50% consistent with the inventive concept and the purposes of the ormamide present invention (e.g., the use of the Surrogate codons to R RNA:RNA <50 TR; 4xSSC TR; 4xSSC enhance expression). 0104. In making Such changes, any techniques known to 0098. The hybrid length is that anticipated for the hybrid persons of skill in the art are utilized. For example, without ized region(s) of the hybridizing polynucleotides. When intending to be limited thereto, the hydropathic index of hybridizing a polynucleotide to a target polynucleotide of amino acids can be considered, as described below with unknown sequence, the hybrid length is assumed to be that of regard to the recombinant proteins and polypeptides of the the hybridizing polynucleotide. When polynucleotides of present invention. The importance of the hydropathic amino US 2009/006925.6 A1 Mar. 12, 2009 acid index in conferring interactive biologic function on logical equivalents. Suitable biological equivalents exhibit polypeptides is generally understood in the art. Kyte et al. the ability to (1) elicitantibodies; (2) react with the surface of 1982. J. Mo1. Bio. 157:105-132. homologous strains and/or heterologous strains; (3) confer 0105. According to further implementations of the inven protection against a live challenge; and/or (4) prevent colo tion, the polynucleotides comprise a polynucleotide library, nization. such as a cDNA library. The preparation of such a library of 0110 Suitable biological equivalents have at least about polynucleotides is well known to persons of skill in the art. A 60% to about 100% similarity to one of the polypeptides person skilled in the art could readily prepare such a library in specified herein (i.e., the even numbered SEQID NOS: 2-6 or accordance with an embodiment of the present invention, amino acid sequences encoded by any of odd numbered SEQ using well known techniques and based upon the guidance ID NOS:1-5 or any of SEQ ID NOS: 12-16), provided the provided herein. As described in further detail below, the equivalent is capable of eliciting Substantially the same polynucleotides of the invention are used in any Suitable immunogenic properties as one of the proteins of this inven context. Such as in vectors, immunogenic compositions, tion. therapeutic compositions, recombinant cells and cell lines, 0111 Alternatively, the biological equivalents have sub assays, kits, tools, etc., as would be well understood by per stantially the same immunogenic properties of one of the sons skilled in the art. proteins in the even numbered SEQ ID NOS: 2-6 or amino acid sequences encoded by any of odd numbered SEQ ID Proteins and Polypeptides NOS:1-5 or any of SEQID NOS: 12-16. According to certain 0106 The present invention also provides recombinant embodiments of the present invention, the biological equiva proteins or polypeptides encoded by the modified polynucle lents have the same immunogenic properties as the even otides of the invention described herein. For example, in numbered SEQ ID NOS 2-6 or amino acid sequences certain embodiments, a recombinant polypeptide or protein encoded by any of odd numbered SEQID NOS:1-5 or any of of the invention is a recombinant that is identical to the ref SEQID NOS: 12-16. erence sequence of even numbered SEQ ID NOS: 2-6 or 0112 The biological equivalents are obtained by generat amino acid sequences encoded by any of odd numbered SEQ ing variants and modifications to the proteins of this inven ID NOS:1-5 or any of SEQ ID NOS: 12-16 (which are tion. These variants and modifications to the proteins are sequences modified in accordance with the invention), that is, obtained by altering the amino acid sequences by insertion, 100% identical, or it may include a number of amino acid deletion or substitution of one or more amino acids. The alterations as compared to the reference sequence such that amino acid sequence is modified, for example by Substitution the percent identity is less than 100%. Such alterations in order to create a polypeptide having Substantially the same include at least one amino acid deletion, Substitution, includ or improved qualities. In a particular embodiment, a means of ing conservative and non-conservative Substitution, or inser introducing alterations comprises making predetermined tion. The alterations occur at the amino- or carboxy-terminal mutations of the nucleic acid sequence of the polypeptide by positions of the reference polypeptide sequence or anywhere site-directed mutagenesis. between those terminal positions, interspersed either indi 0113 Modifications and changes can be made in the struc vidually among the amino acids in the reference amino acid ture of a polypeptide of the present invention while retaining sequence or in one or more contiguous groups within the functional equivalency (such as immunogenicity, therapeutic reference amino acid sequence. benefit, binding affinity, etc). Such modifications and changes 0107 Thus, the invention also provides proteins having are fully contemplated by the present invention. For example, sequence identity to an amino acid sequence of the invention, without limitation, certain amino acids can be substituted for (e.g. even numbered SEQ ID NOS: 2-6 or proteins encoded other amino acids, including nonconserved and conserved by any of odd numbered SEQID NOS:1-5 or any of SEQID Substitution, in a sequence without appreciable loss of func NOS:12-16). Depending on the particular sequence, the tionality/utility (e.g., immunogenicity, therapeutic benefit, degree of sequence identity is greater than 60% (e.g., 60%, etc.). The present invention contemplates any changes to the 70%, 80%, 85%, 90%, 94%, 95%, 97%, 98%, 99%, 99.9% or structure of the polypeptides herein, as well as the nucleic more). These homologous proteins include mutants and acid sequences encoding said polypeptides, wherein the allelic variants. polypeptide retains its functionality or a biologically equiva 0108. In certain embodiments of the invention, the pro lent functionality. teins or polypeptides (e.g., immunological portions and bio 0114. In making Such changes, any techniques known to logical equivalents) generate antibodies. Specifically, the persons of skill in the art may be utilized. For example, antibodies to the polypeptides protect from a challenge. Such without intending to be limited thereto, the hydropathic as intranasal. In further preferred embodiments, the polypep index, hydrophilicity, and the like, of amino acids are consid tides exhibit such protection for homologous strains and at ered (Kyte etal. 1982.J. Mol. Bio. 157:105-132, U.S. Pat. No. least one heterologous strain. The polypeptide may be 4,554,101). selected from even numbered SEQ ID NOS: 2-6 or amino 0115 Biological equivalents of a polypeptide are also pre acid sequences encoded by any of odd numbered SEQ ID pared using site-specific mutagenesis. Site-specific mutagen NOS:1-5 or any of SEQID NOS: 12-16, or the polypeptide esis is a technique useful in the preparation of second genera may be any immunological fragment or biological equivalent tion polypeptides, or biologically functional equivalent of the listed polypeptides. According to an embodiment, the polypeptides or peptides, derived from the sequences thereof, polypeptide is selected from any of the even numbered SEQ through specific mutagenesis of the underlying DNA. Such ID NOS: 2-6 or amino acid sequences encoded by any of odd changes are desirable where amino acid Substitutions are numbered SEQID NOS:1-5 or any of SEQID NOS: 12-16. desirable. The technique further provides a ready ability to 0109. In certain embodiments, the invention relates to prepare and test sequence variants, for example, incorporat allelic or other variants of the polypeptides, which are bio ing one or more of the foregoing considerations, by introduc US 2009/006925.6 A1 Mar. 12, 2009

ing one or more nucleotide sequence changes into the DNA. 0119) “Variant” as the term is used herein, is a polynucle Site-specific mutagenesis allows the production of mutants otide or polypeptide that differs from a reference polynucle through the use of specific oligonucleotide sequences which otide or polypeptide respectively, but retains essential prop encode the DNA sequence of the desired mutation, as well as erties. A typical variant of a polynucleotide differs in a Sufficient number of adjacent nucleotides, to provide a nucleotide sequence from another, reference polynucleotide. primer sequence of sufficient size and sequence complexity to Changes in the nucleotide sequence of the variant may or may form a stable duplex on both sides of the deletion junction not alter the amino acid sequence of a polypeptide encoded by being traversed. Typically, a primer of about 17 to 25 nucle the reference polynucleotide. Nucleotide changes may result otides in length is used, with about 5 to 10 residues on both in amino acid substitutions, additions, deletions, fusions and sides of the junction of the sequence being altered. truncations in the polypeptide encoded by the reference 0116. In general, the technique of site-specific mutagen sequence. A typical variant of a polypeptide differs in amino esis is well known in the art. As will be appreciated, the acid sequence from another, reference polypeptide. Gener technique typically employs a phage vector which can existin ally, differences are limited so that the sequences of the ref both a single stranded and double stranded form. Typically, erence polypeptide and the variant are closely similar overall site-directed mutagenesis in accordance herewith is per and, in many regions, identical (i.e., biologically equivalent). formed by first obtaining a single-stranded vector which A variant and reference polypeptide may differinamino acid includes within its sequence a DNA sequence which encodes sequence by one or more Substitutions, additions, deletions in all or a portion of the polypeptide sequence selected. An any combination. A Substituted or inserted amino acid residue oligonucleotide primer bearing the desired mutated sequence may or may not be one encoded by the genetic code. A variant is prepared (e.g., synthetically). This primer is then annealed of a polynucleotide or polypeptide may be a naturally occur to the single-stranded vector, and extended by the use of ring such as an allelic variant, or it may be a variant that is not enzymes such as E. coli polymerase I Klenow fragment, in known to occur naturally. Non-naturally occurring variants of order to complete the synthesis of the mutation-bearing polynucleotides and polypeptides may be made by mutagen strand. Thus, a heteroduplex is formed wherein one strand esis techniques or by direct synthesis. encodes the original non-mutated sequence and the second I0120 “Identity, as known in the art, is a relationship strand bears the desired mutation. This heteroduplex vector is between two or more polypeptide sequences or two or more then used to transform appropriate cells Such as E. coli cells polynucleotide sequences, as determined by comparing the and clones are selected which include recombinant vectors sequences. In the art, “identity also means the degree of bearing the mutation. Commercially available kits come with sequence relatedness between polypeptide or polynucleotide all the reagents necessary, except the oligonucleotide prim sequences, as the case may be, as determined by the match CS. between strings of such sequences. “Identity” and “similar 0117 The polypeptides of the invention include any pro ity can be readily calculated by known methods, including tein or polypeptide comprising Substantial sequence similar but not limited to those described in Computational Molecu ity and/or biological equivalence to a protein having an amino lar Biology, Lesk, A. M., ed., Oxford University Press, New acid sequence of any of the proteins of the embodiments of York, 1988: Biocomputing: Informatics and Genome the invention such as any of even numbered SEQID NOS 2-6 Projects, Smith, D. W., ed., Academic Press, New York, 1993: or proteins encoded by any of odd numbered SEQID NOS: Computer Analysis of Sequence Data, Part I, Griffin, A. M., 1-5 and 12-16. In addition, the polypeptides of the invention and Griffin, H. G., eds. Humana Press, N.J., 1994: Sequence are not limited to a particular source. Also, the polypeptides Analysis in Molecular Biology, Von Heinje, G., Academic can be prepared recombinantly using any Such technique in Press, 1987; and Sequence Analysis Primer, Gribskov, M. and accordance with the purpose of the invention as described Devereux, J., eds., MStockton Press, New York, 1991; and herein, as is well within the skill in the art, based upon the Carillo, H., and Lipman, D., SIAM.J. Applied Math., 48:1073 guidance provided herein, or in any other synthetic manner, as (1988). Preferred methods to determine identity are designed known in the art. to give the largest match between the sequences tested. Meth 0118. In certain embodiments, a polypeptide is cleaved ods to determine identity and similarity are codified in pub into fragments for use in further structural or functional licly available computer programs. Preferred computer pro analysis, or in the generation of reagents such as related gram methods to determine identity and similarity between polypeptides and specificantibodies. This is accomplished by two sequences include, but are not limited to, the GCG pro treating purified or unpurified polypeptides with a proteolytic gram package (Devereux, J., etal 984), BLASTP. BLASTN, enzyme (i.e., a proteinase) including, but not limited to, serine and FASTA (Altschul, S. F., et al., 1990). The BLASTX pro proteinases (e.g., chymotrypsin, trypsin, plasmin, elastase, gram is publicly available from NCBI and other sources thrombin, Substilin) metal proteinases (e.g., carboxypepti (BLAST Manual, Altschul, S., et al., NCBI NLM NIH dase A, carboxypeptidase B. leucine aminopeptidase, ther Bethesda, Md. 20894; Altschul, S., et al., 1990). The well molysin, collagenase), thiol proteinases (e.g., papain, brome known Smith Waterman algorithm may also be used to deter lain, Streptococcal proteinase, clostripain) and/or acid mine identity. proteinases (e.g., pepsin, gastricsin, trypsinogen). Polypep I0121. In certain embodiments, a polypeptide of the inven tide fragments are also generated using chemical means Such tion (e.g. any of the even numbered SEQID NOS:2-6) com as treatment of the polypeptide with cyanogen bromide prises modifications such as a mature processed form of a (CNBr), 2-nitro-5-thiocyanobenzoic acid, isobenzoic acid, protein, lipidation, glycosylation, de-O-acylation, phospho BNPA-skatole, hydroxylamine or a dilute acid solution. In rylation and the like. other embodiments, the polypeptide fragments of the inven I0122. In one particular embodiment, the polypeptides and tion are recombinantly expressed or prepared via peptide nucleic acids encoding such polypeptides are used in immu synthesis methods known in the art (Barany et al., 1997: U.S. nogenic compositions for preventing or ameliorating infec Pat. No. 5,258.454). tion. US 2009/006925.6 A1 Mar. 12, 2009

0123. The proteins of the invention, including the amino of providing to said mammal an immunogenic composition acid sequences of even numbered SEQ ID NOS: 2-6, their of this invention. The immunogenic composition is a compo fragments, and analogs thereof, or cells expressing them, are sition which is antigenic in the treated mammal Such that an also used as immunogens to produce antibodies immunospe immunologically effective amount of the polypeptide(s) con cific for the polypeptides of the invention. tained in Such composition brings about the desired immune response against infection. Certain embodiments relate to a Antigens method for the treatment, including amelioration, or preven 0124. In certain embodiments, an immunogenic composi tion of infection in a human comprising administering to a tion, including proteins, polynucleotides and equivalents of human an immunologically effective amount of the compo the present invention, is administered as a sole active immu sition. nogen or alternatively, the composition includes other active 0.130. The phrase “immunologically effective amount, as immunogens and/or therapeutics, including other immuno used herein, refers to the administration of that amount to a genic polynucleotides, polypeptides, or immunologically-ac mammalian host (e.g., a human), either in a single dose or as tive proteins of one or more other microbial pathogens (e.g. part of a series of doses, Sufficient to at least cause the immune virus, prion, bacterium, or fungus, without limitation) or cap system of the individual treated to generate a response that Sular polysaccharide. The compositions may comprise one or reduces the clinical impact of the bacterial or viral infection. more desired proteins, fragments or pharmaceutical com This may range from a minimal decrease in bacterial or viral pounds as desired for a chosen indication. In the same man burden to prevention of the infection. Ideally, the treated ner, the compositions of this invention which employ one or individual will not exhibit the more serious clinical manifes more nucleic acids in the composition may also include tations of the bacterial or viral infection. The dosage amount nucleic acids which encode the same diverse group of pro varies depending upon specific conditions of the individual. teins, as noted above. In certain embodiments, a modified This amount is determined in routine trials or otherwise by polynucleotide of the invention comprises a plasmidora viral means known to those skilled in the art. Vector. I0131 The phrase “therapeutically effective amount', as 0125. Any antigen, multi-antigen or multi-valent immu used herein, refers to the administration of that amount to a nogenic composition is contemplated by the present inven mammalian host (e.g., a human), either in a single dose or as tion. For example, the compositions of the present invention part of a series of doses, Sufficient to at least generate a comprise a single protein, combinations of two or more pro response that reduces the impact of the pathogen on the host. teins, one or more polysaccharides, a combination of one or The dosage amount can vary depending on the specific con more proteins, and one or more polysaccharides or any com ditions of the host. The amount is determined through routine bination thereof. Persons of skill in the art would be readily testing or otherwise as known to persons skilled in the art. able to formulate such immunogenic or therapeutic compo I0132) Another specific aspect of the present invention sitions. relates to using as the composition a vector or plasmid which 0126 The present invention also contemplates multi-im expresses a protein of this invention, or an immunogenic or munization (e.g., a prime/boost regimen) or therapeutic regi therapeutic portion thereof. Accordingly, a further aspect of mens wherein any composition useful against a pathogen the invention provides a method of inducing a desired may be combined therein ortherewith the compositions of the response, e.g., immunogenic, in a mammal, which comprises present invention. For example, without limitation, a mam providing to a mammala Vector or plasmid expressing at least malian Subject is administered an immunogenic composition one isolated polypeptide. The protein of the present invention of the present invention and another composition, as part of a is delivered to the mammal using a live, or live attenuted multi-drug regimen. Persons of skill in the art would be vectors. In certain embodiments, the virus is attenuated and readily able to select compositions for use in conjunction with comprises a modified polynucleotide encoding a bacterial the immunogenic and/or therapeutic compositions of the protein, viral protein and the like, containing the genetic present invention for the purposes of developing and imple material necessary for the expression of the polypeptide or menting multi-drug regimens. immunogenic portion as a foreign polypeptide. 0127 Specific embodiments of this invention relate to the use of one or more polypeptides of this invention, or nucleic Viral and Non-Viral Vectors acids encoding such, in a composition or as part of a treatment I0133. The present invention also provides vectors com regimen for the prevention or amelioration of infection. One prising the polynucleotides of the present invention. Accord can combine the polypeptides or polynucleotides with any ing to various embodiments of the invention, vectors are used immunogenic composition for use against infection. One can to transport recombinants of the invention to site of expres also combine the polypeptides or polynucleotides with any sion (e.g., transcription, translation/protein synthesis). Thus, other protein or polysaccharide-based immunogenic compo the vectors are used in vivo or in vitro depending upon the sition. desired objective. Any suitable vectors for accomplishing the 0128. In certain embodiments, the polypeptides, frag objectives consistent with the inventive concept are contem ments and equivalents are used as part of a conjugate immu plated by the present invention. nogenic composition; wherein one or more proteins or 0.134 Viral vectors such as lentiviruses, retroviruses, her polypeptides are conjugated to a carrier protein in order to pes viruses, adenoviruses, adeno-associated viruses, vaccinia generate a composition that has immunogenic properties virus, baculovirus, and other recombinant viruses with desir against several serotypes and/or against several diseases. able cellular tropism, are particularly useful for cellular Alternatively, one of the polypeptides is used as a carrier assays in vitro and in vivo. Thus, a nucleic acid encoding a protein for other immunogenic polypeptides. protein or immunogenic fragment thereof can be introduced 0129. The present invention also relates to a method of in vivo, ex vivo, or in vitro using a viral vector or through inducing immune responses in a mammal comprising the step direct introduction of DNA. Expression in targeted tissues US 2009/006925.6 A1 Mar. 12, 2009

can be effected by targeting the transgenic vector to specific use of vectors derived from the AAVs for transferring genes in cells, such as with a viral vector or a receptor ligand, or by vitro and in vivo has been described (see, PCT Publication using a tissue-specific promoter, or both. Targeted gene deliv Nos. WO 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797, ery is described in PCT Publication No. WO95/28494, which 368 and 5,139,941; European Publication No. EP 488 528). is incorporated herein by reference in its entirety. The replication defective recombinant AAVs according to the 0135 Viral vectors commonly used for in vivo or ex vivo invention can be prepared by cotransfecting a plasmid con targeting and therapy procedures include DNA vectors and taining the nucleic acid sequence of interest flanked by two RNA vectors. Methods for constructing and using viral vec AAV inverted terminal repeat (ITR) regions, and a plasmid tors are known in the art (e.g., Miller and Rosman, BioTech carrying the AAV encapsidation genes (rep and cap genes), niques, 1992, 7:980-990). In certain embodiments, the viral into a cell line which is infected with a human helper virus vectors are replication-defective, that is, they are unable to (for example an adenovirus). The AAV recombinants which replicate autonomously in the target cell. In other embodi are produced are then purified by standard techniques. ments, the viral vector is a live attenuated virus. In one par 0.139 vectors. In another implementation of ticular embodiment, the replication defective virus is a mini the present invention, the nucleic acid can be introduced in a mal virus, i.e., it retains only the sequences of its genome retroviral vector, e.g., as described in U.S. Pat. No. 5,399.346; which are necessary for encapsulating the genome to produce Mannet al., Cell, 1983,33:153: U.S. Pat. Nos. 4,650,764 and viral particles. 4,980,289; Markowitzet al., J. Virol, 1988, 62:1120; U.S. Pat. 0.136 Various companies produce viral vectors commer No. 5,124,263: European Publication Nos. EP 453242 and cially, including, but not limited to, Avigen, Inc. (Alameda, EP178 220; Bernstein et al., Genet. Eng., 1985, 7:235: Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; ret McCormick, BioTechnology, 1985, 3:689; PCT Publication roviral, adenoviral, AAV vectors, and lentiviral vectors), No. WO95/07358; and Kuo et al., Blood, 1993, 82:845, each Clontech (retroviral and baculoviral vectors), Genovo, Inc. of which is incorporated by reference in its entirety. The (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec (ad retroviruses are integrating viruses that infect dividing cells. enoviral vectors), IntroGene (Leiden, Netherlands; adenovi The retrovirus genome includes two LTRS, an encapsidation ral vectors), Molecular Medicine (retroviral, adenoviral, sequence and three coding regions (gag, pol and enV). In AAV, and herpes viral vectors), Norgen (adenoviral vectors), recombinant retroviral vectors, the gag, poland envgenes are Oxford BioMedica (Oxford, United Kingdom; lentiviral vec generally deleted, in whole or in part, and replaced with a tors), and Transgene (Strasbourg, France; adenoviral, vac heterologous nucleic acid sequence of interest. These vectors cinia, retroviral, and lentiviral vectors), incorporated by ref can be constructed from different types of retrovirus, such as, erence herein in its entirety. HIV. MoMuIV (“murine Moloney leukaemia virus'), MSV 0.137 Adenovirus vectors. Adenoviruses are eukaryotic (“murine Moloney sarcoma virus), HaSV (“Harvey sarcoma DNA viruses that can be modified to efficiently deliver a virus); SNV (“spleen necrosis virus); RSV (“Rous sarcoma nucleic acid of this invention to a variety of cell types. Various virus) and Friend virus. Suitable packaging cell lines have serotypes of adenovirus exist. In one particular embodiment, been described in the prior art, in particular the cell line an adenovirus (Ad) is a type 2, type 4, type 5, or type 7 human PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (PCT adenoviruses (Ad 2, Ad 4, Ad 5 or Ad 7) or adenoviruses of Publication No. WO 90/02806) and the GP+enVAm-12 cell animal origin (see PCT Publication No. WO 94/26914). line (PCT Publication No. WO 89/07150). In addition, the Those adenoviruses of animal origin which can be used recombinant retroviral vectors can contain modifications within the scope of the present invention include adenovi within the LTRS for Suppressing transcriptional activity as ruses of canine, bovine, murine (e.g., Mavl, Beard et al., well as extensive encapsidation sequences which may include Virology, 1990, 75-81) bovine, porcine, avian, and simian a part of the gag gene (Bender et al., J. Virol, 1987, 61: 1639). (e.g., SAV) origin. In one embodiment, the adenovirus of Recombinant retroviral vectors are purified by standard tech animal origin is a canine adenovirus, such as a CAV2 aden niques known to those having ordinary skill in the art. ovirus (e.g., Manhattan or A26/61 strain, ATCC VR-800). 0140. Retroviral vectors can be constructed to function as Various replication defective adenovirus and minimum aden infectious particles or to undergo a single round of transfec ovirus vectors have been described (PCT Publication Nos. tion. In the former case, the virus is modified to retain all of its WO94/26914, WO95/02697, WO 94/28938, WO 94/28152, genes except for those responsible for oncogenic transforma WO 94/12649, WO95/02697, WO 96/22378). The replica tion properties, and to express the heterologous gene. Non tion defective recombinant adenoviruses according to the infectious viral vectors are manipulated to destroy the viral invention can be prepared by any technique known to the packaging signal, but retain the structural genes required to person skilled in the art (Levrero et al., Gene, 1991, 101: 195; package the co-introduced virus engineered to contain the European Publication No. EP 185573; Graham, EMBO.J., heterologous gene and the packaging signals. Thus, the viral 1984, 3:2917: Graham et al., J. Gen. Virol., 1977, 36:59). particles that are produced are not capable of producing addi Recombinant adenoviruses are recovered and purified using tional virus. standard molecular biological techniques, which are well 0141 Retrovirus vectors can also be introduced by DNA known to persons of ordinary skill in the art. viruses, which permits one cycle of retroviral replication and 0138 Adeno-associated viruses. The adeno-associated amplifies transfection efficiency (see PCT Publication Nos. viruses (AAV) are DNA viruses of relatively small size that WO 95/22617, WO 95/26411, WO 96/39036 and WO can integrate, in a stable and site-specific manner, into the 97/19182). genome of the cells which they infect. They are able to infect 0.142 Lentivirus vectors. In another implementation of the a wide spectrum of cells without inducing any effects on present invention, lentiviral vectors are used as agents for the cellular growth, morphology or differentiation, and they do direct delivery and Sustained expression of a transgene in not appear to be involved in human pathologies. The AAV several tissue types, including brain, retina, muscle, liver and genome has been cloned, sequenced and characterized. The blood. The vectors efficiently transduce dividing and nondi US 2009/006925.6 A1 Mar. 12, 2009 viding cells in these tissues, and effect long-term expression some. An assay is used to confirm that the polynucleotides of the gene of interest. For a review, see, Naldini, Curr. Opin. administered by immunization do not give rise to a trans Biotechnol., 1998,9:457-63; see also Zufferey, et al., J. Virol., formed phenotype in the host (U.S. Pat. No. 6,168.918). 1998, 72:9873-80). Lentiviral packaging cell lines are avail 0149. It is also possible to introduce the vector in vivo as a able and known generally in the art. They facilitate the pro naked DNA plasmid. Naked DNA vectors for vaccine pur duction of high-titer lentivirus vectors for gene therapy. An poses or gene therapy can be introduced into the desired host example is a tetracycline-inducible VSV-G pseudotyped len cells by methods known in the art, e.g., electroporation, tivirus packaging cell line that can generate virus particles at microinjection, cell fusion, DEAE dextran, calcium phos titers greater than 106 IU/mL for at least 3 to 4 days (Kafri, et phate precipitation, use of a gene gun, or use of a DNA vector al., J. Virol, 1999, 73: 576-584). The vector produced by the transporter (e.g., Wu etal, J. Biol. Chem., 1992,267:963-967: inducible cell line can be concentrated as needed for effi Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624; Cana ciently transducing non-dividing cells in vitro and in vivo. dian Patent Application No. 2,012.311; Williams et al., Proc. 0143. In another implementation of the present invention, Natl. Acad. Sci. USA, 1991, 88:2726-2730). Receptor-medi a modified polynucleotide of the invention is delivered via ated DNA delivery approaches can also be used (Curiel et al., Mononegavirales. Viruses of the Order Mononegavirales are Hum. Gene Ther, 1992, 3:147-154: Wu and Wu, J. Biol. non-segmented, negative dtranded RNA viruses (e.g., Chem., 1987, 262:4429-4432). U.S. Pat. Nos. 5,580,859 and described in U.S. Pat. No. 6,033,886, incorporated herein by 5,589.466 disclose delivery of exogenous DNA sequences, reference) free of transfection facilitating agents, in a mammal. More 0144. In one particular embodiment, a modified poly recently, a relatively low voltage, high efficiency in vivo DNA nucleotide of the invention is delivered via Vesicular Stoma transfer technique, termed electrotransfer, has been described titis Virus (VSV). Genetically modified VSV strains, attenu (Miret al., C. P. Acad. Sci., 1988, 321:893; PCT Publication ating VSV mutations and VSV rescue methods are well Nos. WO99/01157; WO99/01158; WO99/01175). Accord known in theart, e.g. see U.S. Pat. Nos. 6,033,886: 6,168,943: ingly, additional embodiments of the present invention relates 6,596,529. to a method of inducing an immune response in a human 0145 Non-viral vectors. In another implementation of the comprising administering to said human an amount of a DNA present invention, the vector can be introduced in vivo by molecule encoding a polypeptide of this invention, optionally lipofection, as “naked DNA, or with other transfection with a transfection-facilitating agent, where said polypeptide, facilitating agents (peptides, polymers, etc.). Synthetic cat when expressed, retains the desired functionality and, when ionic lipids are used to prepare liposomes for in vivo trans incorporated into an immunogenic composition and admin fection of a gene encoding a marker (Felgner, et. al., Proc. istered to a human, provides protection without inducing Natl. Acad. Sci. U.S.A., 1987, 84:7413-7417; Felgner and enhanced disease upon Subsequent infection of the human Ringold, Science, 1989, 337:387-388; see Mackey, et al., with a pathogen. Transfection-facilitating agents are known Proc. Natl. Acad. Sci. U.S.A., 1988, 85:8027-8031: Ulmer et in the art and include bupivicaine, and other local anesthetics al., Science, 1993, 259:1745-1748). Useful lipid compounds (for examples see U.S. Pat. No. 5,739,118) and cationic and compositions for transfer of nucleic acids are described in polyamines (as published in International Patent Application PCT Patent Publication NoS. WO 95/18863 and WO WO96/10038), which are hereby incorporated by reference. 96/17823, and in U.S. Pat. No. 5.459,127. Lipids may be 0150. According to an embodiment of the present inven chemically coupled to other molecules for the purpose of tion, the IL-15 constructs as described herein are adminis targeting (see Mackey, et al. Supra). Targeted peptides, e.g., tered in a plasmid. According to an embodiment, the plasmid hormones or neurotransmitters, and proteins such as antibod of the present invention comprises SEQID NOS: 18, 19, 20 or ies, or non-peptide molecules could be coupled to liposomes combinations thereof. The preparation of plasmids is well chemically. known in the art. A person of ordinary skill in the art could 0146. Other molecules are also useful for facilitating readily prepare a plasmid having the modified polynucle transfection of a nucleic acid in Vivo, Such as a cationic otide, Such as the IL-15 constructs, for example, in accor oligopeptide (e.g., PCT Patent Publication No. WO dance with the present invention, based upon the guidance 95/21931), peptides derived from DNA binding proteins provided herein. For example, the preparation of plasmids is (e.g., PCT Patent Publication No. WO 96/25508), or a cat described in U.S. Pat. No. 5,593.972, which is incorporated ionic polymer (e.g., PCT Patent Publication No. WO by reference in its entirety. 95/21931). Adjuvants 0147 In certain embodiments, a polynucleotide modified for optimal expression in a mammalian host (i.e., comprising 0151. According to an embodiment of the present inven Surrogate codons) is administered directly to the host as an tion, the polynucleotides of the present invention may be used immunogenic composition. The polynucleotide is introduced as adjuvants, for example, as adjuvants for vaccines, such as directly into the host either as “naked' DNA (U.S. Pat. No. DNA and/or RNA vaccines. Techniques for the preparation of 5,580,859) or formulated in compositions with agents which adjuvants, DNA vaccines and RNA vaccines are well known facilitate immunization, such as bupivicaine and other local in the art. A person of skill in the art would readily be able to anesthetics (U.S. Pat. No. 5,593.972) and cationic prepare an adjuvant, DNA vaccine and/or RNA vaccine and polyamines (U.S. Pat. No. 6,127,170). the like, using the embodiments of the present invention, 0148. In this polynucleotide immunization procedure, the based upon the guidance provided herein. polypeptides of the invention are expressed on a transient 0152 The present invention contemplates that the modi basis in vivo; no genetic material is inserted or integrated into fied polynucleotides of the present invention may be used the chromosomes of the host. This procedure is to be distin alone or in combination with other compounds or composi guished from gene therapy, where the goal is to insert or tions for any desired effect. For example, the modified poly integrate the genetic material of interest into the chromo nucleotides of the present invention may be administered in US 2009/006925.6 A1 Mar. 12, 2009

combination with a DNA and/or RNA vaccine or as part of the Such as wetting or emulsifying agents, preservatives or buff DNA and/or RNA vaccine (e.g., as part of a plasmid contain ers, which enhance the shelf life or effectiveness of the anti ing the DNA and/or RNA vaccine). The modified polyncle body. The preparation and use of pharmaceutically accept otides of the present invention may be administered sepa able carriers is well known in the art. Except insofar as any rately but contemporaneously with the administration of the conventional media or agent is incompatible with the active DNA and/or RNA vaccine, include administering during, ingredient, use thereof in the compositions of the present before or after. Further, the polynucleotides of the present invention is contemplated. invention may be administered alone. 0157 An immunogenic composition of the invention is 0153 Exemplary DNA vaccines with which the present formulated to be compatible with its intended route of admin invention may be combined in any manner include, without istration. Examples of routes of administration include limitation, nucleotides coding for the Plasmodium (malarial parenteral (e.g., intravenous, intradermal, Subcutaneous, agent) proteins such as Pfalciparum, P. vivax, P malariae, intramuscular, intraperitoneal), mucosal (e.g., oral, rectal, and P ovale CSP: SSP2(TRAP); Pfs 16 (Sheba); LSA-1; intranasal, buccal, vaginal, respiratory) and transdermal LSA-2: LSA-3; STARP; MSA-1 (MSP-1, PMMSA, PSA, (topical). Other modes of administration employ oral formu p185, p190); MSA-2 (MSP-2, Gymmsa, gp56, 38-45 kDa lations, pulmonary formulations, Suppositories, and transder antigen); RESA (Pfl55); EBA-175: AMA-1 (Pfs3); SERA mal applications, for example, without limitation. Oral for (p113, p.126, SERP, Pfl40); RAP-1; RAP-2: RhopH3; mulations, for example, include Such normally employed PfHRP-II; Pf55; Pf35; GBP (96-R); ABRA (p.101); Exp-1 excipients as, for example, pharmaceutical grades of manni (CRA, Ag5.1); Aldolase; Duffy binding protein of P. vivax: tol, lactose, starch, magnesium Stearate, sodium saccharine, Reticulocyte binding proteins; HSP70-1 (p75); Pfg25; Pfg28; cellulose, magnesium carbonate, and the like, without limi Pfg48/45; and Pfg230. DNA and RNA vaccines also may tation. comprise nucleotides coding for proteins associated with the 0158. The present invention contemplates the use of GP or NP genes from the ebola virus; and the HPV6a L2. embodiments of the invention as adjuvants or co-adjuvants, HPV6a E1, HPV6a E2, HPV6a E4, HPV6a E5, HPV6a E6, for example, as adjuvants to DNA or RNA vaccines/immu and HPV6a E7 proteins from the human Papillomavirus 6a nogenic composition. The immunogenic compositions of the (HPV6a). According to an embodiment, the DNA and RNA invention can include one or more adjuvants, or be adminis vaccines code for HIV proteins, including, but not limited to, tered along with one or more adjuvants, including, but not the glycoproteins gp41, gp120, gp140, and gp160; and pro limited to aluminum salts (alum) Such as aluminum phos teins encoded by the gag (the proteins p55, p39, p24, p17 and phate and aluminum hydroxide, Mycobacterium tuberculo p15), env, rev, tat, nef, vpr, vpx, prot, and pol (the proteins sis, Bordetella pertussis, bacterial lipopolysaccharides, ami p66/p51 and p31-34) genes found in HIV. noalkyl glucosamine phosphate compounds (AGP), or 0154 According to an embodiment of the present inven derivatives or analogs thereof, which are available from tion, the IL-15 constructs of the present invention (e.g., SEQ Corixa (Hamilton, Mont.), and which are described in U.S. ID NOS:12-16) is used in combination with DNA and/or Pat. No. 6,113,918; one such AGP is 2-((R)-3-Tetradecanoy RNA vaccine. e.g., a DNA vaccine against HIV/AIDS. loxytetradecanoylaminoethyl 2-Deoxy-4-O-phosphono-3- According to an embodiment, SEQ ID NO:14 is used (e.g., O—(R)-3-tetradecanoyoxytetradecanoyl-2-((R)-3-tetrade administered contemporaneously and/or combined in a plas canoyoxytetradecanoylamino-b-D-glucopyranoside, which mid or other vector or composition) in combination with a is also known as 529 (formerly known as RC529), which is DNA vaccine against HIV/AIDS. formulated as an aqueous form or as a stable emulsion, MPLTM (3-O-deacylated monophosphoryl lipid A) (Corixa) Compositions described in U.S. Pat. No. 4.912,094, synthetic polynucle 0155 One aspect of the present invention provides com otides such as oligonucleotides containing a CpG motif (U.S. positions, such as immunogenic compositions and therapeu Pat. No. 6,207,646), polypeptides, saponins such as Quil A or tic compositions, etc., which comprise a modified polynucle STIMULONTM QS-21 (Antigenics, Framingham, Mass.), otide of the present invention, a protein or polypeptide described in U.S. Pat. No. 5,057,540, a pertussis toxin (PT), encoded by said recombinant polynucleotide, an antibody to an E. coli heat-labile toxin (LT), particularly LT-K63, LT said protein or polypeptide, or the like, including any combi R72, CT-S109, PT-K9/G129; see, e.g., International Patent nations thereof. For example, compositions that have the Publication Nos. WO 93/13302 and WO92/19265, cholera ability to confer protection against a live challenge and/or toxin (either in a wild-type or mutant form, e.g., wherein the prevent colonization are contemplated by the present inven glutamic acid at amino acid position 29 is replaced by another tion. amino acid, such as a histidine, in accordance with published 0156 The formulation of such compositions is well International Patent Application number WO 00/18434). known to persons skilled in this field. Compositions of the 0159 Various cytokines and lymphokines are suitable for invention, according to an embodiment, include a pharma use as adjuvants. One Such adjuvant is granulocyte-macroph ceutically acceptable carrier. Suitable pharmaceutically age colony stimulating factor (GM-CSF), which has a nucle acceptable carriers and/or diluents include any and all con otide sequence as described in U.S. Pat. No. 5,078,996. A ventional Solvents, dispersion media, fillers, Solid carriers, plasmid containing GM-CSF cDNA has been transformed aqueous solutions, coatings, antibacterial and antifungal into E. coli and has been deposited with the American Type agents, isotonic and absorption delaying agents, and the like. Culture Collection (ATCC), 1081 University Boulevard, Suitable pharmaceutically acceptable carriers include, for Manassas, Va. 20110-2209, under Accession Number 39900. example, one or more of water, saline, phosphate buffered The cytokine Interleukin-12 (IL-12) is another adjuvant saline, dextrose, glycerol, ethanol and the like, as well as which is described in U.S. Pat. No. 5,723,127. Other cytok combinations thereof. Pharmaceutically acceptable carriers ines or lymphokines have been shown to have immune modu may further comprise minor amounts of auxiliary Substances lating activity, including, but not limited to, the interleukins US 2009/006925.6 A1 Mar. 12, 2009

1-C, 1-B, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16, 17 and 18, the 0.165. The description of the specific embodiments will so interferons-C, Bandy, granulocyte colony Stimulating factor, fully reveal the general nature of the invention that others can, and the tumor necrosis factors C. and B, and are Suitable foruse by applying knowledge within the skill of the art, readily as adjuvants. modify and/or adapt for various applications such specific 0160. In certain embodiments, the proteins of this inven embodiments, without undue experimentation, without tion are used in a composition for oral administration which departing from the general concept of the present invention. includes a mucosal adjuvant and used for the treatment or Therefore, Suchadaptations and modifications are intended to prevention of infection in a mammalian host (e.g., a human). be within the meaning and range of equivalents of the dis The mucosal adjuvant can be a wild-type cholera toxin or, a closed embodiments, based on the teaching and guidance derivative of a cholera holotoxin, wherein the A subunit is presented herein. It is to be understood that the phraseology or mutagenized or chemically modified. For a specific cholera terminology herein is for the purpose of description and not of toxin which may be particularly useful in preparing immu limitation, such that the terminology or phraseology of the nogenic compositions of this invention, see the mutant chol present specification is to be interpreted by the skilled artisan era holotoxin E29H, as disclosed in Published International in light of the teachings and guidance presented herein, in Application WO 00/18434, which is hereby incorporated combination with the knowledge of one of ordinary skill in herein by reference in its entirety. These may be added to, or the art. A person skilled in the art would know, or be able to conjugated with, the polypeptides of this invention. The same ascertain, using no more than routine experimentation, many techniques are applied to other molecules with mucosal adju equivalents to the specific embodiments of the invention vant or delivery properties such as Escherichia coli heat labile described herein, based upon the guidance provided herein. toxin (LT). Other compounds with mucosal adjuvant or deliv 0166 The following examples are included to demon ery activity may be used Such as bile; polycations such as strate particular embodiments of the invention. However, DEAE-dextran and polyornithine; detergents such as sodium those of skill in the art should, in view of the present disclo dodecyl benzene Sulphate; lipid-conjugated materials; anti Sure, appreciate that many changes can be made in the specific biotics such as Streptomycin; vitamin A; and other com embodiments which are disclosed and still obtain a like or pounds that alter the structural or functional integrity of similar result without departing from the spirit and scope of mucosal Surfaces. Other mucosally active compounds the invention. The following examples are offered by way of include derivatives of microbial structures such as MDP; illustration and are not intended to limit the invention in any acridine and cimetidine. STIMULONTM QS-21, MPL, and way. IL-12, as described above, may also be used. EXAMPLES 0161 The compositions of this invention may be delivered in the form of ISCOMS (immune stimulating complexes), Example 1 ISCOMS containing CTB, liposomes or encapsulated in Enhancement of HPV 16 E7 expression compounds such as acrylates or poly(DL-lactide-co-glyco 0.167 a. One example of a “modified’ polynucleotide side) to form microspheres of a size Suited to adsorption. The sequence demonstrating "enhanced' levels of protein expres proteins of this invention may also be incorporated into oily sion is shown below in SEQ ID NO:1. The modified poly emulsions. nucleotide's sequence incorporates Surrogate codons encod 0162 Recombinant cells, recombinant cell lines, assays ing the 98 amino acid human papillomavirus (HPV)16 E7 and kits that provide or use same and the like are also con protein sequence (e.g., see HPV 16 Accession No. K02718 in templated by the present invention. A person skilled in the art NCBI database). would readily understand how to prepare and use Such 0.168. The enhanced sequence of the polynucleotide in embodiments of the present invention, based upon the guid accordance with an embodiment of the invention is deter ance provided herein. mined by selecting Suitable Surrogate codons. Surrogate 0163 The present invention also relates to an antibody, codons were selected in order to alter the A and T (or A and U which may either be a monoclonal or polyclonal antibody, in the case of RNA) content of the naturally-occurring (wild specific for polypeptides as described above. Such antibodies type) gene. The Surrogate codons are those that encode the may be produced by methods which are well known to those amino acids alanine, arginine, glutamic acid, glycine, isoleu skilled in the art. cine, leucine, proline, serine, threonine, and Valine. Accord 0164. According to a further implementation of the ingly, the modified nucleic acid sequence had surrogate present invention, a method is provided for diagnosing a codons for each of these amino acids throughout the condition in a mammal comprising: detecting the presence of sequence. For the remaining 11 amino acids, no alterations immune complexes in the mammal or a tissue sample from were made, thereby leaving the corresponding naturally-oc said mammal, said mammal or tissue sample being contacted curring codons in place. with an antibody composition comprising antibodies that 0169. The modified sequence may be determined manu immunospecifically bind with at least one polypeptide com ally or by computer-assisted methods. As such, the informa prising the amino acid sequence of any of the even numbered tion technology, including hardware, Software, algorithms, SEQID NOS: 2-6; wherein the mammal or tissue sample is arrays, databases and the like, directed to the determination of contacted with the antibody composition under conditions the modified sequences of the present invention are contem suitable for the formation of the immune complexes. plated herein.

SEQ ID NO: 1 (polynucleotide) and SEQ ID NO:2 (protein) 1. ATGCATGGGGATACGCCTACGCTCCATGAATATATGCTCGATCTCCAACCTGA 1. M H G D T P T L H E Y M L D L Q. P E US 2009/006925.6 A1 Mar. 12, 2009 18

- Continued

54 GACGACGGATCTCTACTGTTATGAGCAACT CAATGACAGCTCCGAGGAGGAGG 18 T T D L Y C Y E O L N D S S E E E

1. Of ATGAAATTGATGGGCCTGCGGGGCAAGCGGAACCTGACCGGGCCCATTACAAT 36 D E I D G P A G O A E, P D R A H Y N

160 ATTGTCACCTTTTGTTGCAAGTGTGACTCCACGCTCCGGCTCTGCGTCCAAAG 54 I W T F C C K C D S T L R L C V Q S

213 CACGCACGTCGACATTCGGACGCTCGAAGACCTGCTCATGGGCACGCTCGGGA 71. T H W D I R T L E D L. L. M G T L G

266 TTGTGTGCCCCATCTGTTCCCAGAAACCTTAATAG 89 I W C P I C S O K P

(0170 Referring to SEQID NO:1 above, the recombinant tors. All HPV 16 E7 genes were cloned into pcDNA3.1 (Invit nucleotide sequence of HPV 16 E7 (Accession No. K02718) rogen, Carlsbad, Calif.). While a variety of different trans incorporates Surrogate codons but retains the capacity to fecting agents could be utilized, the experiments listed herein encode the wild type E7 protein. were performed using Lipofectamine (invitrogen) according (0171 b. The nucleic acid sequence of SEQID NO:1 was to manufacturer's instructions. Total cell lysates were har assembled from oligonucleotides that were 100 nucleotides vested 48 hours after transfection in SDS-sample buffer con in length and corresponding in polarity to the positive (sense) taining 1% SDS and 2-mercaptoethanol. Equivalent amounts Strand sequence shown above. A person of skill in the art of each transfectant lysate were loaded and electrophoresed would readily be able to select suitable oligonucleotides on 4-20% tris-glycine gradient SDS-polyacrylamide gels. depending upon the desired sequence in accordance with the HPV 16 E7 protein was detected by an E7-specific mono present invention. Suitable oligonucleotides are available clonal antibody (Zymed Laboratories, San Francisco, Calif.). from a variety of commercial vendors, such as InvitrogenTM 0.174. The expression levels of the surrogate codon modi (Carlsbad, Calif.). fied HPV 16 E7 gene (SEQ ID NO:1) were markedly (0172 “Bridge' oligos 50 nucleotides in length and anti enhanced compared to the expression levels of the wild type sense in polarity were designed to straddle the joints at the HPV 16 E7 gene. The expression levels of the surrogate codon ends of each sense 100-mer oligo. This strategy facilitated the modified HPV 16E7 was comparable to the expression level hybridization of 25 nucleotides at the ends of each 100-mer of the “preferred” codon modified HPV 16E7 (data not targeted for ligation. Aheat stable ligase (Ampligase, Epicen shown). tre, Wis.) was used at 68°C. to ligate the 100-mer sense oligos together. The entire open reading frame (for HPV 16 E7. Example 2 approximately 300 nucleotides) was then PCR amplified Enhancement of HIV-1 Gag p37 Expression using oligos corresponding to the 5' and 3' boundaries of the 0.175. A second example demonstrating the unexpected ORF. The fidelity of the intended final ORF was verified by results of using “surrogate' codons in lieu of wild-type sequencing reactions. codons in a nucleic acid sequence was found for the HIV-1 0173 This HPV 16E7 gene containing surrogate codons gag gene, specifically the p37 component of the full-length was tested for expression levels by Western blot (data not p55 protein. shown). Rhabdomyosarcoma (RD) cells (American Type 0176 a. The amino acid sequence of the HXB2 strain of Culture Collection, Manassas, Va. ATCC# CCL136) were HIV-1 (NCBI Accession No. K03455) was selected as a rep transfected with the indicated plasmid DNA expression vec resentative HIV-1 gag gene.

SEO ID NO:3 (polynucleotide) and SEQ ID NO: 4 (protein) 1. ATGGGGGCGCGGGCGTCCGTCCTCTCCGGGGGGGAGCTCGATCGGTGGGAGAAA 1. M. G. A. R. A. S W L S. G. G E L D R W E K.

55 ATTCGGCTCCGGCCGGGGGGGAAGAAAAAATATAAACT CAAACATATTGTCTGG 19 I R L R P G. G. K. K. K. Y. K. L. K. H. I. W. W.

109 GCGTCCCGGGAGCTCGAGCGGTTCGCGGTCAATCCGGGGCTGCTCGAGACGTCC 37 A S R E L E R F A. W N P G L L E T S

163 GAGGGCTGTCGGCAAATTCTCGGGCAGCTCCAACCGTCCCTCCAGACGGGGTCC 55 E G C R Q I L G O L Q. P S L Q T G S

217 GAGGAGCTCCGGTCCCTCTATAATACGGTCGCGACGCTCTATTGTGTCCATCAA 73 E E L R S L Y N T W A T L Y C W H Q

271 CGGATTGAGATTAAAGACACGAAGGAGGCGCTCGACAAGATTGAGGAGGAGCAA 91 R. I E I K D T K E A L. D. K. I E E E Q US 2009/006925.6 A1 Mar. 12, 2009 19

- Continued

AACAAATCCAAGAAAAAAGCGCAGCAAGCGGCGGCGGACACGGGGCACTCCAAT N K S K K K A. Q. Q. A. A. A D T G H S N

CAGGTCTCCCAAAATTACCCGATTGTCCAGAACATTCAGGGGCAAATGGTCCAT Q V S O N Y P I V O N I O G O M W H

CAGGCGATTTCCCCGCGGACGCTCAATGCGTGGGTCAAAGTCGTCGAGGAGAAG O A I S P R T L N A W V K V V E E K

GCGTTCTCCCCGGAGGTCATTCCGATGTTTTCAGCGCTCTCCGAGGGGGCGACG A F S P E W II P. M. F. S. A. L S E G A T

CCGCAAGATCTCAACACGATGCTCAACACGGTCGGGGGGCATCAAGCGGCGATG P O D L N T M L N T W G G H Q A A M

CAAATGCTCAAAGAGACGATTAATGAGGAGGCGGCGGAGTGGGATCGGGTCCAT O M L K E T I N E E A A E W D R W H

649 CCGGTCCATGCGGGGCCGATTGCGCCGGGGCAGATGCGGGAGCCGCGGGGGTCC 217 P W H A G P I A P G O M R. E. P R G S

7O3 GACATTGCGGGGACGACGTCCACGCTCCAGGAGCAAATTGGGTGGATGACGAAT 235 D I A G T T S T L Q E O I G W M T N

757 AATCCGCCGATTCCGGTCGGGGAGATTTATAAACGGTGGATTATTCTCGGGCTC 253 N P P I P W G E I Y K R W I I L. G. L.

811 AATAAAATTGTCCGGATGTATTCCCCGACGTCCATTCTCGACATTCGGCAAGGG 271 N K I V R. M. Y S P T S I L D I R O G

865 CCCAAGGAGCCGTTTCGGGACTATGTAGACCGGTTCTATAAAACGCTCCGGGCG 289 P K E P. F. R D Y W D R F Y K. T L R A

919 GAGCAAGCGTCCCAGGAGGTCAAAAATTGGATGACGGAGACGCTCCTCGTCCAA 3. Of E Q A S Q E V K N W M T E T L L V Q

973 AATGCGAACCCGGATTGTAAGACGATTCT CAAAGCGCTCGGGCCGGCGGCTACG 3.25 N A N P D C K T I L. K. A. L. G. P. A. A. T.

102.7 CTCGAGGAGATGATGACGGCGTGTCAGGGGGTCGGGGGGCCGGGGCATAAGGCG 343 L E E M M T A C Q G W G G P G H K A

1081 CGGGTCCTCTAA 361 R W I

(0177 Referring to SEQ ID NO:3, an altered nucleotide merase (Pfx Turbo, Invitrogen). This staggered, overlapping sequence of the HXB2 strain of HIV-1 gag gene (Accession arrangement was performed to assemble the entire ~1.1 kb No. K03455) incorporating surrogate codons but retaining gag gene encoding p37. The double stranded but “nicked” the capacity to encode the 363 amino acid wild type p37 assembled gene was then ligated using a heat stable ligase component of the gag protein, was constructed. (Ampligase). 0.178 The HIV-1 gag p37 gene incorporating surrogate codons was assembled by a different method than that used 0180 PCR oligos representing the 5' and 3' most regions for the HPV 16 E7 (Example 1). This gene was assembled of the p37 ORF were then used to amplify the entire gene, using a series of 100-mer sense and antisense oligos contain which was Subsequently cloned into the vector and sequenced ing overlapping 25 nucleotides of sequence as illustrated to confirm the fidelity in assembly of the predicted sequence. below. 0181. The expression levels of a plasmid DNA construct containing the altered/“surrogate'gagp37 gene shown above ATG 3 3 were tested by transfection in Cos7 cells (ATCC CRL 1651). The levels of gag present in the Supernatant 48 hours post 3' . . . . etc. infection was quantified with an ELISA assay using a com (0179. Each 100 mer was phosphorylated () on the 5' end mercially available kit (Coulter p24 kit, Beckman Coulter to facilitate downstream ligation. For reference, the 5' end of catalog #PN6604535). The plasmid construct set forth in the gag gene, containing the initiation codon ATG, is depicted SEQID NO:7 was used for transfection of the wild-type gag (sense oligo); an antisense oligo beneath it was designed to p37. The plasmid construct set forth in SEQ ID NO:8 was contain complementary sequence of 25 nucleotides to facili used for transfection of the recombinant gag gene (modified tate hybridization and subsequent fill in by a DNA poly in accordance with an embodiment of the present invention).

US 2009/006925.6 A1 Mar. 12, 2009 30

- Continued gaga catgaa citgctott to gatgtcacca Caagcataga taaaaagaag alagacagaat 366 O atgcaattitt tgataaactt gatgtaatga atataggtaa tggaagatat acattlactaa 372 O attgta acac cticagt catt acacaggc ct gtc.caaagat gtc.ctittgaa CCaattic Coa 378 O tacattattg taccc.cggct ggittatgcga ttctaaagtg taatgataat aagttcaatg 384 O gaac aggacc atgtacaaat gtcagcacaa tacaatgtac acatggaatt aagc.cagtag 3900 tgtcaactica actgctgtta aatggcagtic tag cagaagg aggagaggta ataattagat 396 O ctgaaaatct cacagacaat gctaaaacca taatagtaca gct caaggaa Cctgtagaaa t caattgtac aag acccaac aacaatacala gaaaaagtat acatatggga cCaggagcag cattt tatgc aagaggagaa gtaataggag atataagaca agcacattgc aac attagta 414 O gaggaagatg gaatgacact ttaaaacaga tagctaaaaa attaa.gagaa Caatittaata 42OO aaacaataag CCttalaccala t cct caggag gggacct aga aattgtaatg Cacacttitta 426 O attgttggagg ggaatttitt c tactgtaata caacacagot gtttaatagt acttggaatg 432O agaatgatac tacctggaat aatacagcag ggtcaaataa caatgaaact at Cacactic C 438 O catgtagaat aaaacaaatt ataalacaggt ggCaggalagt aggaaaagca atgitatgcc c 4 44 O citcc catcag tggaccaatt aattgttitat Caaat at CaC agggct atta ttaacaagag 4500 atggtggtga Caacaataat acaatagaga cct tcagacc tggaggagga gatatgaggg 456 O acaattggag aagtgaatta tataaatata aagtagtaag aattgagc.ca ttaggaatag

CaCC Caccala ggcaaagaga agagtggtgc aaagagaaaa aagagcagtg ggaat aggag 468O citatgttcct tgggttcttg ggagcagcag gaagcactat ggg.cgcagcg t cagtgacgc 474. O tgacgg taca ggc.ca.gacta ttattgttctg gtatagtgca acagoaaaac aatttgctga 48OO gagdtatcga ggcgcaa.cag catctgttgc aact cacagt Ctggggcatc aag cagct cc 486 O aggctagagt Cctggctatg gaaagatacc taaaggat.ca acagotccta gggatttggg 492 O gttgct Ctgg aaaact catt tgcaccacta atgtgccttg gaatgct agt tggagtaata 498O aatctotgga Caagatttgg cataa.catga Cctggatgga gtgggacaga gaaattgaca 5040 attacacaaa. attaatataC accittaattg alagcatcgca gatccagcag gaaaagaatg aacaagaatt attggaattg gatagttggg Caagtttgttg gagttggittt gacat ct caa 516 O aatggctgtg gtatatagga gtatt cataa tagtaatagg aggtttagta ggtttaaaaa 522 O tagtttittgc tgtactitt ct at agtaaata gagittaggca gggat actica CCatt at Cat 528 O ttcagaccc.g cct cocagcc ccgacaggcc cgaaggaatc gaagaaggag 534 O gtggagaga.g agacagagac agat.ccgatc aattagtgac tggattctta gcact catct 54 OO gggacgat ct gcggagcCtg tgcct citt.ca gctaccaccg Cttgagagac ttact cittga 546 O ttgtagcgag gattgttggaa Cttctgggac gCagggggtg ggaagcc ctg aagtattggit 552O ggaatctoct gcaatattgg att Caggaac taaagaatag tgctgttagt ttgcttaacg 558 O ccacagctat agcagtag cc gaggggacag ataggattat agalagtagta Calaaggattg 564 O gtagagct at tot CCaCata Cctagaagaa taalgacaggg Cttagaaagg gctittgctat st OO aatagggcgc gcc.gagct cq citgat cagcc tcgactgtgc cittctagttg c cago catct 576. O gttgtttgcc cct coccc.gt gcct tccttg accctggaag gtgcc acticc cactgtc.citt 582O to Ctaataaa. atgaggaaat tgcatcgcat tgtctgagta ggtgt catt C tattotgggg 588 O US 2009/006925.6 A1 Mar. 12, 2009

- Continued ggtggggtgg gCaggacag Caagggggag gattgggaag acaat agcag gCatgctggg 594 O gaattit 5946

0189 A plasmid map of the plasmid construct set forth in Example 4 SEQID NO:9 is provided as FIG. 4 and a plasmid map of the plasmid construct set forth in SEQID NO:10 is provided as Modification of the Env Gene Increased gp160 Pro FIG.S. tein Levels Relative to Wild-Type 0.190 Western blot detection and ELISA methods were employed to compare transfected cells expressing the wild 0.194. A further study comparing the expression of a modi type or the modified gp160 genes. fied polynucleotide of an embodiment of the present inven 0191 Two Western blots confirmed gp160 antigen speci tion for gp160 to the wild-type version of the gene was con ficity from SEQID NO:9 plasmid construct-transfected 293 ducted. cells forty eight hours later (data not shown). Initial studies 0.195 For the purposes of the study, a modified polynucle tested two SEQID NO:9 plasmid construct clones with later otide of an embodiment of the present invention for gp160 focus on clone 6, hereafter just denoted SEQID NO:9. These was prepared as described in Example 3 above. A wild-type Western blots demonstrated recognition of SEQ ID NO: 9 gp160 polynucleotide for the gene was also obtained for the plasmid construct-transfected lysates by both an anti IIIB study. gp120 polyclonal rabbit serum as well as an anti-MN gp41 0196. Expression of the two types of polynucleotides was monoclonal antibody (data not shown). Each blot revealed measured using the systems described in Examples 1-3 reactivity with their respective positive control recombinant above. proteins (451 for gp160 and MN expressed in E. coli for gp41. (0197) Referring to FIG. 1, the results of the study are Since the amino acid sequences differ between the 6101 illustrated by the graph. As is clearly shown, the modified primary isolate (encoded by the SEQID NO:9 plasmid con polynucleotide of an embodiment of the present invention for struct) and the MN strain, no direct quantitative comparisons the gp160 ('optimized”) gene provides substantially better can be made between these envelopes in these Western blots expression than the wild-type gene. or in the ELISA assays listed below. 0.192 Enhanced expression levels of the 6101 gp160 enve Example 5 lope gene according to an embodiment of the present inven tion was observed. The plasmid construct for the gene modi Enhanced Expression of Human IL-15 fied in accordance with an embodiment of the present invention (SEQID NO:9) expressed substantially higher lev 0198 A study was conducted to compare IL-15 expres els of gp160 compared to the wild-type 6101 gene (which was sion by various IL-15 constructs in accordance with embodi undetectable by Western blot). Envelope 6101 gp160 expres ments of the present invention, Such as an IL-15 recombinant construct (modified with Surrogate codons) with a human IgE sion levels were quantified for 293 as well as for COS-7, Hela, leader sequence or with the long leader sequence, unmodified and RD cell lines after transient transfection from total cell IL-15 with an IgE leader, and two alternative optimized IL-15 lysates using an anti-gp120 ELISA capture kit (ABI, Cat No. constructs with IgE leader against expression by other IL-15 15-102-000). constructs. The results of the study show that the constructs of the present invention provide unexpectedly improved expres TABLE IV sion of IL-15. In particular, the IgE leader sequence in com bination with the less intensive modified Surrogate codon HIV-1 Gp160 6101 protein levels (in ng/ml) from total cell lysates approach provides synergistically improved expression over Cells currently used IL-15 constructs and comparable results to codon optimized or “preferred codon approaches with a Constructs COS-7 Hela RD 293 lower intensive and thus highly efficient and accurate construct for modified polynucleotide 4 5.4 O.8 8O approach. The experimental procedures and results are (SEQID NO:9) described below and illustrated in the following Tables and in construct for wild-type :::::: :::::: :::::: :::::: FIGS. 6-10. (SEQID NO: 10) 0199 Various constructs were used for comparative pur * Lower limit of standard curve = 78 pg/ml poses, as follows: **not detected 0200) 1. IL-15 constructs with the native IL-15 signal pep 0193 From these studies it can be concluded that the con tide replaced by the human IgE leader sequence. struct for the modified gene (SEQ ID NO: 9) expresses the 0201 2. IL-15 constructs with optimized codons (codon altered 6101 gp160 protein at levels far superior (almost 100 optimization alternative 1. times) to its wild-type counterpart (SEQID NO:10) in several 0202. 3. IL-15 constructs with the IL-15 nucleotide cell lines (as shown in Table IV). Quantification of this pri sequence optimized to reduce mRNA secondary structure mary isolate can be achieved by an ABI anti-gp120 ELISAkit (codon optimization alternative 2). and is at substantially lower levels than observed for p37 gag 0203 4. IL-15 constructs with combinations of IgE leader (in the ug/ml range in cell lysates). sequences and gene optimization techniques. US 2009/006925.6 A1 Mar. 12, 2009 32

Cloning: Results: 0204 All gene sequences were designed based upon pub Human IL-15 Constructs: lished codon tables and synthesized from Blue Heron Tech nologies. Genes were then subcloned into the DNA vaccine 0208. The following seven human IL-15 inserts were sub vector backbone. cloned into a vector backbone, which contains human CMV promoter. All the constructs were confirmed by sequencing Cell Culture and Transfection: and used for in vitro and in vivo human IL-15 expression assayS. 0205 RD, 293, Hela and COS-7 cells were used in tran sient transfections. All transfections were carried out using Fugene-6 (Roche) according to the manufacturer's instruc tions. A total of 0.25 mg of human IL-15 plasmid and 0.5 mg ------LP-IL-15-IgE leader (surrogate codons) of SEAP (a secreted form of human placental alkaline phos ...... Current clinical IL-15 (native IL-15 with long phatase) control vector with 4 ml of Fugene-6 was used for signal peptide) each transfection. For dose titration, 0.25-2.0 mg of the test ------Native IL-15 with IL-15-IgE leader that replaces plasmid was used along with the control DNA and the total the long signal peptide DNA was made up to a final concentration of 2.0 mg per ------O-IL-15-IgE leader (preffered codons) transfection. Dose titration was performed to identify an ------BH-IL-15-IgE leader (secondary structure appropriate concentration of plasmid to be used for compara optimization) tive analysis. Forty-eight hours after transfection, cell culture O-15 with a long signal peptide media and cells were harvested and analyzed for IL-15 by LP-15 with a long signal peptide ELISA (R&D Systems) and CTLL2 proliferation assay. The RNA optimization with a long signal peptide cell lysates were tested for total protein concentration by Micro BCA protein assay. Data is depicted as pg of IL-15 per Native Leader Sequence mg of protein in cell lysates and pg of IL-15 per 10,000 units +++++ IgE Leader Sequence of seap activity. 0209. As shown in Table V(A) and V(B), constructs according to embodiments of the present invention signifi Intramuscular Immunization of Mice: cantly improve IL-15 expression in vitro. In particular, Table 0206 Six to eight-week-old female BALB/c mice were V(A) shows expression in cells and supernatants of 293 cells. used in this study. Each group consisted of 2 animals and mice Table V(B) shows expression in cells and supernatants of RD were immunized intramuscularly in both quadriceps muscles cells with a total of 200 mg plasmid DNA (formulated with 0.25% bupivacaine) in a 50 ml Volume using a 28-gauge needle. In all 4 muscles were analysed at each time point. The quadri ceps muscles were taken at 2, 5, 9 and 15 days post-immuni (A) zation and homogenized in cell lysis buffer (50 mM Tris, Human IL-15 expression in 293 cell lysates (ELISA) pH8.0-50 mM NaCl-1% Triton-X100) containing proteinase Fold increase inhibitor mixture (Roche). The cell lysates were subjected to human IL15 (pg/mg compared to three freeze and thaw cycles, centrifuged and Supernatants Group protein) WLV125M were evaluated for IL-15 protein by ELISA (R&D Systems). WLV125M 7139.83 1.OO Data are represented as average expression in 4 muscle WLV134M 23893.23 3.35 samples per group. WLV186M 1230O2.31 17.23 WLV187M 80523.75 11.28 WLV188M 29772.71 4.17 CTLL2 Cell Proliferation Assay WLV211M 33OOO.66 4.62 WLV217M 11403.65 1.60 0207 Mouse CTLL2 cells were washed twice with PBS WLV225M 29.103.13 4.08 and incubated in a 96 well-plate at a density of 100000 cells/ WLVOO1 AM O.OO O well in complete medium with either different amounts of Human IL-15 expression in 293 cell Supernatants (ELISA) human recombinant IL-15 (R&D Systems) as standard con trols or indicated media of cells transfected with hL-15 human IL15 Fold increase expression construct. Forty eight hours post-incubation, (pg/ml/10000 unit compared to MTT reagent (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltet Group SEAP) WLV125M razolium bromide) was added and further incubated for four WLV125M 64.24 1.OO WLV134M 928.76 14.46 hours. Conversion of the tetrazolium salt to the purple forma WLV186M 68.07.04 105.96 Zon product by mitochondrial enzymes in viable cells allows WLV187M 4.389.32 68.33 a visual assessment of the reaction. When the purple forma WLV188M 1327.20 20.66 Zon precipitate was clearly visible in the microscope the cells WLV211M 967.94 15.07 WLV217M 217.81 3.39 were lysed with the detergent and absorbances read at 570 WLV225M 1556.50 24.23 nm. Final concentration is based upon the known standards WLVOO1 AM O.OO O used in the assay and data are represented as pg of IL-15 per ml of Supernatant from transfected cells. US 2009/006925.6 A1 Mar. 12, 2009 33

(B) Human IL-15 expression in Human IL-15 expression RD cell Supernatants (ELISA in RD cell lysates (ELISA Fold human IL15 Fold increase increase (pg/ml/10000 unit compared to human IL15 (pg/mg compared to Group SEAP) WLV125M Group protein) WLV125M 72.97 1.OO WLV12SM 1056.64 WLV134M S28.40 7.24 WLV134M 2786.32 WLV186M 9544.01 130.79 WLV186M 20877.53 1 WLV187M 4102.73 56.22 WLV187M 7287.57 WLV188M 1548.02 21.21 WLV188M 3275.43 WLV211M 6287.93 86.17 WLV211M 6183.53 WLV217M 4O7.16 5.58 WLV217M 1409.34 WLV225M 1958.41 26.84 WLV22SM 4443.84 WLVOO1 AM O.OO O WLVOO1 AM O.OO

0210 Table VI shows in vivo gene expression from IL-15 expression. Replacement of native IL-15 signal peptide constructs in accordance with the invention as well as previ sequence with that of human IgE leader up-regulated its ously used IL-15 constructs for purposes of comparison. expression by 5-8 fold demonstrating the negative regulatory Codon engineering in addition to the replacement of the feature of the IL-15 leader. Not only did optimized further native signal peptide with human IgE leader significantly enhance the expression by 4-15 fold, but even more supris improved IL-15 expression in vivo. Four mice per group ingly, the less intensive Surrogate codon approach as received 200 mg of plasmid DNA. Animals were sacrificed described herein did so as well. and analyzed at 2, 5, 9 and 15 days after immunization. Data 0213 Codon engineering in addition to secretary signal Summarized are an average IL-15 protein expression from a Substitution resulted in as much as 40-100 fold increase in group of 4 muscles per time point. IL-15 gene expression in various cell lines tested. The func tionality of IL-15 produced from constructs was demon strated by CTLL2 cell proliferation assay. 0214 Consistent with in vitro data, in vivo gene expres Human IL-15 expression in the mouse sion from the IL-15 constructs according to embodiments of muscles(pg. 10 mg of protein the invention was considerably elevated. Taken together, this Groups Day 2 Day 5 Day 9 Day 15 data Suggest that this combined method represents a novel WLV125M 2.959 2.714 2.889 O.845 and unexpected approach for enhancing IL-15 gene expres WLV134M 4.134 3.028 2.927 O.811 S1O. WLV186M 25.846 31.830 3.403 1.220 0215. The IgE leader sequence for use in certain embodi WLV187M 15.072 4.826 2.499 O.829 ments of the invention is provided below. 0211 Table VII shows the results of the CTLL2 assay. IgE Leader Sequence (SEQ ID NO: 11) Supernatants from RD cells transfected with optimized con 0216 structs induced 5-30 fold higher functional IL-15 than the native plasmid in a MTT cell proliferation bioassay (see materials and methods for details). The proliferation rate was ATGGATTGGACTTGGATCTTATTTTTAGTTGCTGCGCTACTAGAGTTCA estimated from a standard curve obtained with purified recombinant human IL-15 (pg/ml). TTCT 0217. The following are the nucleic acid sequences of constructs in accordance with embodiments of the present Human IL-15 expression in 293 cell lysates (CTLL2ASSay invention. Leader sequences are indicated by underlining. Fold increase human IL15 (ngi ml of compared to Surrogate codon usage HulL-15 sequence Group Supernatant) WLV125M (SEQ ID NO:12) AIGCGGATTTCCAAACCTCATCTCAGGTCCATTTCCATCCAGTGCTACCT WLV125M 3.12 1.OO WLV134M 16.22 5.19 CTGTCTCCTCCTCAACTCCCATTTTCTCACGGAAGCTGGCATTCATGTCT WLV186M 98.95 31.69 WLV187M 71.42 22.87 TCATTGTCGGCTGTTTCTCCGCGGGGCTCCCTAAAACGGAAGCCAACTGG WLV188M 34.36 11.01 WLVOO1 AM O.OO O.OO GTGAATGTCATTTCCGATCTCAAAAAAATTGAAGATCTCATTCAATCCAT

GCATATTGATGCGACGCTCTATACGGAATCCGATGTCCACCCCTCCTGCA 0212. The foregoing study demonstrates that various gene modification strategies significantly improve human IL-15 US 2009/006925.6 A1 Mar. 12, 2009 34

- Continued - Continued AAGTCACCGCGATGAAGTGCTTTCTCCTCGAGCTCCAAGTCATTTCCCTC TCTGGTTGTAAAGAATGCGAAGAACTTGAAGAAAAAAATATAAAAGAATT

GAGTCCGGGGATGCGTCCATTCATGATACGGTCGAAAATCTGATCATCCT TCTGCAATCATTTGTCCACATCGTTCAAATGTTTATCAATACCTCTTAG

CGCGAACAACTCCCTCTCCTCCAATGGGAATGTCACGGAATCCGGGTGCA The following is the sequence of naturally occurring human IL-15 sequence provided herein for AAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTCTCCAGTCC comparative purposes.

TTTGTCCATATTGTCCAAATGTTCATCAACACGTCCTAG Human IL-15 sequence (SEQ ID NO:17) AGAGAATCGAAACCACATTGAGAAGTATTTCCACCAGGCTAC IgE leader Human IL-15 sequence (SEQ ID NO: 13) ATGGATTGGACTTGGATCTTATTTTTAGTTGCTGCGCTACTAGAGTTCA GTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCT

TTCTAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTA TCATTTTGGGCTGTTTCAGGCAGGGCTCCAAAACAGAAGCCAACTGG

TTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCAC GTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTAT

CCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGT GCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCA

TATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATC AAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTT

TGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAA GAGTCTGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCT

TCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATT AGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCA

TTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA AAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGT

IgE leader + surrogate codon usage HulL-15 TTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA sequence (SEQ ID NO:14) ATGGATTGGACGTGGATCCTCTTTCTCGTCGCGGCGGCGACGCGGGTCCA The following is the nucleic acid sequence for the O-IL-15 - IgE leader plasmid construct TTccAACTGGGTGAATGTCATTTCCGATCTCAAAAAAATTGAAGATCTCA (SEQ ID NO:18): AAATGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATA TTCAATCCATGCATATTGATGCGACGCTCTATACGGAATCCGATGTCCAC CCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGT CCCTCCTGCAAAGTCACCGCGATGAAGTGCTTTCTCCTCGAGCTCCAAGT TGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGC CATTTCCCTCGAGTCCGGGGATGCGTCCATTCATGATACGGTCGAAAATC TTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTT TGATCATCCTCGCGAACAACTCCCTCTCCTCCAATGGGAATGTCACGGAA CAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGT TCCGGGTGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATT CAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGCGTTCA TCTCCAGTCCTTTGTCCATATTGTCCAAATGTTCATCAACACGTCCTAG AAATGGTATGCGTTTTGACACATCCACTATATATCCGTGTCGTTCTGTCC IgE leader + optimized HulL-15 sequence (optimized alternative 1) (SEQ ID NO: 15 ACTCCTGAATCCCATTCCAGAAATTCTCTAGCGATTCCAGAAGTTTCTCA ATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCCGCCACCCGCGTGCA GAGTCGGAAAGTTGACCAGACATTACGAACTGGCACAGATGGTCATAACC CTCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGA TGAAGGAAGATCTGATTGCTTAACTGCTTCAGTTAAGACCGACGCGCTCG TCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCAC TCGTATAACAGATGCGATGATGCAGACCAATCAACATGGCACCTGCCATT CCCAGCTGCAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGT GCTACCTGTACAGTCAAGGATGGTAGAAATGTTGTCGGTCCTTGCACACG GATCAGCCTGGAGAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACC AATATTACGCCATTTGCCTGCATATTCAAACAGCTCTTCTACGATAAGGG TGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAG CACAAATCGCATCGTGGAACGTTTGGGCTTCTACCGATTTAGCAGTTTGA AGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTT TACACTTTCTCTAAGTATCCACCTGAATCATAAATCGGCAAAATAGAGAA CCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTAG AAATTGACCATGTGTAAGCGGCCAATCTGATTCCACCTGAGATGCATAAT IgE leader + Secondary structure optimized HulL-15 sequence (Optimized Alternative 2) (SEQ ID NO: 16) CTAGTAGAATCTCTTCGCTATCAAAATTCACTTCCACCTTCCACTCACCG ATGGATTGGACCTGGATCCTCTTTCTTGTCGCCGCTGCCACTCGAGTACA GTTGTCCATTCATGGCTGAACTCTGCTTCCTCTGTTGACATGACACACAT TTcAAACTGGGTAAATGTGATTTCCGACCTTAAAAAAATTGAAGACCTTA CATCTCAATATCCGAATACGGACCATCAGTCTGACGACCAAGAGAGCCAT TCCAAAGCATGCACATAGACGCCACCCTTTATACTGAATCCGACGTACAC AAACACCAATAGCCTTAACATCATCCCCATATTTATCCAATATTCGTTCC CCCTCCTGCAAAGTTACCGCCATGAAATGTTTTCTCCTCGAACTCCAAGT TTAATTTCATGAACAATCTTCATTCTTTCTTCTCTAGTCATTATTATTGG AATTAGCCTCGAATCCGGAGACGCCTCTATCCACGACACAGTTGAAAACC TCCGTTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTA TCATAATCCTTGCAAATAACTCTCTTAGCTCAAACGGAAATGTTACTGAA TTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTT

US 2009/006925.6 A1 Mar. 12, 2009 37

- Continued - Continued

AGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCT CAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAAT

TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAA GACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGA

ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTC CTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG

TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTT TGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCA

CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG

GCCTACATAC CTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCAT

GCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGAT TGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGA

AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT GCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTT

GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAG TGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGCGCGCGT

AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC CGACCACCATGGATTGGACCTGGATCCTCTTTCTTGTCGCCGCTGCCACT

GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC CGAGTACATTCAAACTGGGTAAATGTGATTTCCGACCTTAAAAAAATTGA

CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC AGACCTTATCCAAAGCATGCACATAGACGCCACCCTTTATACTGAATCCG

GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGC ACGTACACCCCTCCTGCAAAGTTACCGCCATGAAATGTTTTCTCCTCGAA

AACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACAT CTCCAAGTAATTAGCCTCGAATCCGGAGACGCCTCTATCCACGACACAGT

GTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCT TGAAAACCTCATAATCCTTGCAAATAACTCTCTTAGCTCAAACGGAAATG

TTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG TTACTGAATCTGGTTGTAAAGAATGCGAAGAACTTGAAGAAAAAAATATA

TCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTAC AAAGAATTTCTGCAATCATTTGTCCACATCGTTCAAATGTTTATCAATAC

GCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCT CTCTTAGTGAGTCGACGGGCGACGCGAAACTTGGGCCCACTCGAGAGGCG

GCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTG CGCCGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC

GAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGG TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC

CTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGC CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGT

TGCTTCGCGATGTACGGGCCAGATATAGCCGCGGCATCGATGATATCCAT AGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGG

TGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGT AGGATTGGGAAGACAATAGCAGGCATGCTGGGGAATTT CCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTA 0218. The invention now being fully described, it will be ATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC from the spirit or scope of the invention as set forth herein. The foregoing describes the preferred embodiments of the CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGAC present invention along with a number of possible alterna TTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG tives. These embodiments, however, are merely for example and the invention is not restricted thereto.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 2O

<210 SEQ ID NO 1 <211 LENGTH: 3 OO &212> TYPE: DNA <213> ORGANISM: Human papillomavirus &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (294)

<4 OO SEQUENCE: 1

US 2009/006925.6 A1 Mar. 12, 2009 40

- Continued

34 O 345 35. O gtC g g g g CC9 g Cat aag gC9 C9g gtC Ct c taa 1092 Val Gly Gly Pro Gly. His Lys Ala Arg Val Lieu. 355 360

<210 SEQ ID NO 4 <211 LENGTH: 363 &212> TYPE: PRT <213> ORGANISM: Human immunodeficiency virus type 1

<4 OO SEQUENCE: 4 Met Gly Ala Arg Ala Ser Val Lieu. Ser Gly Gly Glu Lieu. Asp Arg Trp 1. 5 1O 15 Glu Lys Ile Arg Lieu. Arg Pro Gly Gly Lys Llys Llys Tyr Lys Lieu Lys 2O 25 3 O His Ile Val Trp Ala Ser Arg Glu Lieu. Glu Arg Phe Ala Wall Asn Pro 35 4 O 45 Gly Lieu. Lieu. Glu Thir Ser Glu Gly Cys Arg Glin Ile Lieu. Gly Glin Lieu. SO 55 60 Glin Pro Ser Lieu. Glin Thr Gly Ser Glu Glu Lieu. Arg Ser Lieu. Tyr Asn 65 70 7s 8O Thr Val Ala Thir Lieu. Tyr Cys Val His Glin Arg Ile Glu Ile Lys Asp 85 90 95 Thr Lys Glu Ala Lieu. Asp Llys Ile Glu Glu Glu Gln ASn Lys Ser Lys 1OO 105 11 O Llys Lys Ala Glin Glin Ala Ala Ala Asp Thr Gly His Ser Asn Glin Val 115 12 O 125 Ser Glin Asn Tyr Pro Ile Val Glin Asn Ile Glin Gly Gln Met Val His 13 O 135 14 O Glin Ala Ile Ser Pro Arg Thr Lieu. Asn Ala Trp Val Llys Val Val Glu 145 150 155 160 Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser 1.65 17O 17s Glu Gly Ala Thr Pro Glin Asp Lieu. Asn Thr Met Lieu. Asn Thr Val Gly 18O 185 19 O Gly His Glin Ala Ala Met Gln Met Lieu Lys Glu Thir Ile Asin Glu Glu 195 2OO 2O5 Ala Ala Glu Trp Asp Arg Val His Pro Wal His Ala Gly Pro Ile Ala 21 O 215 22O Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly. Thir Thr 225 23 O 235 24 O Ser Thr Lieu. Glin Glu Glin Ile Gly Trp Met Thr Asn Asn Pro Pro Ile 245 250 255 Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Lieu. Gly Lieu. Asn Lys 26 O 265 27 O Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Lieu. Asp Ile Arg Glin Gly 27s 28O 285 Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Lieu. 29 O 295 3 OO Arg Ala Glu Glin Ala Ser Glin Glu Val Lys Asn Trp Met Thr Glu Thr 3. OS 310 315 32O Lieu. Lieu Val Glin Asn Ala Asn Pro Asp Cys Llys Thir Ile Lieu Lys Ala 3.25 330 335

US 2009/006925.6 A1 Mar. 12, 2009 44

- Continued

835 84 O 845 gtc. cag cqg gcg Ctic ctic taatga 2568 Val Glin Arg Ala Lieu. Lieu. 850

<210 SEQ ID NO 6 <211 LENGTH: 854 &212> TYPE: PRT <213> ORGANISM: Human immunodeficiency virus type 1

<4 OO SEQUENCE: 6 Met Arg Ala Lys Glu Met Arg Llys Ser Cys Glin His Lieu. Arg Llys Trp 1. 5 1O 15 Gly Ile Lieu. Lieu. Phe Gly Val Lieu Met Ile Cys Ser Ala Glu Glu Lys 2O 25 3 O Lieu. Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 4 O 45 Thir Thr Lieu. Phe Cys Ala Ser Asp Ala Lys Ala His His Ala Glu Ala SO 55 60 His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 7s 8O Glin Glu Val Ile Lieu. Glu Asn Val Thr Glu Lys Tyr Asn Met Trp Llys 85 90 95 ASn Asn Met Val Asp Gln Met His Glu Asp Ile Ile Ser Lieu. Trp Asp 1OO 105 11 O Gln Ser Leu Lys Pro Cys Val Lys Lieu. Thr Pro Leu. Cys Val Thr Lieu. 115 12 O 125 Asn Cys Thr Asn Ala Thr Tyr Thr Asn Ser Asp Ser Lys Asn Ser Thr 13 O 135 14 O Ser Asn. Ser Ser Lieu. Glu Asp Ser Gly Lys Gly Asp Met Asn. Cys Ser 145 150 155 160 Phe Asp Val Thir Thr Ser Ile Asp Llys Llys Llys Llys Thr Glu Tyr Ala 1.65 17O 17s Ile Phe Asp Llys Lieu. Asp Wal Met Asn. Ile Gly Asn Gly Arg Tyr Thr 18O 185 19 O Lieu. Lieu. Asn. Cys Asn Arg Ser Val Ile Thr Glin Ala Cys Pro Llys Met 195 2OO 2O5 Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Tyr Ala 21 O 215 22O Ile Lieu Lys Cys Asn Asp Asn Llys Phe Asin Gly Thr Gly Pro Cys Thr 225 23 O 235 24 O Asn Val Ser Thr Ile Glin Cys Thr His Gly Ile Llys Pro Val Val Ser 245 250 255 Thr Glin Lieu. Lieu. Lieu. Asn Gly Ser Lieu Ala Glu Gly Gly Glu Val Ile 26 O 265 27 O Ile Arg Ser Glu Asn Lieu. Thir Asp Asn Ala Lys Thir Ile Ile Val Glin 27s 28O 285 Lieu Lys Glu Pro Val Glu Ile Asn. Cys Thr Arg Pro Asn. Asn. Asn Thr 29 O 295 3 OO Arg Llys Ser Ile His Met Gly Pro Gly Ala Ala Phe Tyr Ala Arg Gly 3. OS 310 315 32O Glu Val Ile Gly Asp Ile Arg Glin Ala His Cys Asn. Ile Ser Arg Gly 3.25 330 335 US 2009/006925.6 A1 Mar. 12, 2009 45

- Continued

Arg Trp Asn Asp Thir Lieu Lys Glin Ile Ala Lys Llys Lieu. Arg Glu Glin 34 O 345 35. O Phe Asn Llys Thir Ile Ser Lieu. Asn Glin Ser Ser Gly Gly Asp Lieu. Glu 355 360 365 Ile Val Met His Thr Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn 37 O 375 38O Thir Thr Gln Leu Phe Asn Ser Thr Trp Asin Glu Asn Asp Thir Thir Trp 385 390 395 4 OO Asn Asn Thr Ala Gly Ser Asn Asn Asn Glu Thir Ile Thr Lieu Pro Cys 4 OS 41O 415 Arg Ile Lys Glin Ile Ile Asn Arg Trp Glin Glu Val Gly Lys Ala Met 42O 425 43 O Tyr Ala Pro Pro Ile Ser Gly Pro Ile Asin Cys Lieu Ser Asn Ile Thr 435 44 O 445 Gly Lieu. Lieu. Lieu. Thir Arg Asp Gly Gly Asp Asn. Asn. Asn. Thir Ile Glu 450 45.5 460 Thir Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu 465 470 47s 48O Lieu. Tyr Lys Tyr Llys Val Val Arg Ile Glu Pro Lieu. Gly Ile Ala Pro 485 490 495 Thir Lys Ala Lys Arg Arg Val Val Glin Arg Glu Lys Arg Ala Val Gly SOO 505 51O Ile Gly Ala Met Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met 515 52O 525 Gly Ala Ala Ser Val Thir Lieu. Thr Val Glin Ala Arg Lieu. Lieu. Lieu. Ser 53 O 535 54 O Gly Ile Val Glin Glin Glin Asn. Asn Lieu. Lieu. Gly Ala Ile Glu Ala Glin 5.45 550 555 560 Glin His Lieu. Lieu. Glin Lieu. Thr Val Trp Gly Ile Lys Glin Lieu. Glin Ala 565 st O sts Arg Val Lieu Ala Met Glu Arg Tyr Lieu Lys Asp Glin Gln Lieu. Lieu. Gly 58O 585 59 O Ile Trp Gly Cys Ser Gly Lys Lieu. Ile Cys Thr Thr Asn Val Pro Trp 595 6OO 605 Asn Ala Ser Trp Ser Asn Llys Ser Lieu. Asp Llys Ile Trp His Asn Met 610 615 62O Thir Trp Met Glu Trp Asp Arg Glu Ile Asp Asn Tyr Thr Lys Lieu. Ile 625 630 635 64 O Tyr Thr Lieu. Ile Glu Ala Ser Glin Ile Glin Glin Glu Lys Asn. Glu Glin 645 650 655 Glu Lieu. Lieu. Glu Lieu. Asp Ser Trp Ala Ser Lieu. Trp Ser Trp Phe Asp 660 665 67 O Ile Ser Lys Trp Leu Trp Tyr Ile Gly Val Phe Ile Ile Val Ile Gly 675 68O 685 Gly Lieu Val Gly Lieu Lys Ile Val Phe Ala Val Lieu. Ser Ile Val Asn 69 O. 695 7 OO Arg Val Arg Glin Gly Tyr Ser Pro Leu Ser Phe Glin Thr Arg Lieu Pro 7 Os 71O 71s 72O Ala Pro Arg Gly Pro Asp Arg Pro Glu Gly Ile Glu Glu Gly Gly Gly 72 73 O 73 US 2009/006925.6 A1 Mar. 12, 2009 46

- Continued Glu Arg Asp Arg Asp Arg Ser Asp Gln Lieu Val Thr Gly Phe Lieu Ala 740 74. 7 O Lieu. Ile Trp Asp Asp Lieu. Arg Ser Lieu. Cys Lieu. Phe Ser Tyr His Arg 7ss 760 765 Lieu. Arg Asp Lieu Lleu Lieu. Ile Val Ala Arg Ile Val Glu Lieu. Lieu. Gly 770 775 78O Arg Arg Gly Trp Glu Ala Lieu Lys Tyr Trp Trp Asn Lieu. Lieu. Glin Tyr 78s 79 O 79. 8OO Trp Ile Glin Glu Lieu Lys Asn. Ser Ala Val Ser Lieu. Lieu. Asn Ala Thr 805 810 815 Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Ile Ile Glu Val Val Glin 82O 825 83 O Arg Ile Gly Arg Ala Ile Lieu. His Ile Pro Arg Arg Ile Pro Glin Gly 835 84 O 845 Val Glin Arg Ala Lieu. Lieu. 850

<210 SEQ ID NO 7 <211 LENGTH: 4418 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OO SEQUENCE: 7 aaatgggggg gctgaggit ct gcct cqtgaa galaggtgttg Ctgact cata C caggcctga 6 O atcgc.cc.cat catc.ca.gc.ca gaaagtgagg gagcc acggit tatgagagc tittgttgtag 12 O gtggaccagt ttgattitt gaacttittgc tittgccacgg alacggtctgc gttgtcggga 18O agatgcgtga tictdatcc tt caact cagca aaagttctgat ttatt caa.ca aagcc.gc.cgt. 24 O cc.cgtcaagt cagcgtaatg citctgccagt gttacaiacca attaaccaat tctg.cgttca 3OO aaatgg tatg cqttittgaca catccact at at atc.cgtgt cqttctgtcc act cotgaat 360 cc catt coag aaattcticta gcgatt.ccag aagtttct ca gag toggaaa gttgaccaga 42O cattacgaac toggcacagat gigt cataa.cc tdaaggaaga t ctdattgct taactgcttic 48O agittaaga cc gacgc.gct cq t cqtatalaca gatgcgatga tigcagaccala t caac atggc 54 O acctgc catt gctacctgta Cagt caagga tigg tagaaat gttgtcggit C Cttgcacacg 6OO aat attacgc catttgcc td catatt caaa cagct cittct acgataaggg cacaaatcgc 660 atcgtggaac gtttgggctt ctaccgattt agcagtttga tacactittct ctaagtaticc 72 O acctgaatca taaatcggca aaatagagaa aaattgacca ttgtaagcg gccaatctga 78O titccacct ga gatgcataat c tagtagaat ct citt cqcta t caaaattica citt coaccitt 84 O c cactic accqi gttgtc catt catggctgaa citctgct tcc tictdttgaca tdacacacat 9 OO catcto aata t cogaatacg gaccatcagt ctdacgacca agaga.gc.cat aaacaccaat 96.O agccittaa.ca to atcc cc at atttatccaa tatt cqttcc ttaattt cat gaacaat citt 1 O2O cattctittct tctictagt ca ttatt attgg to cqtt cata acacic ccttg tattactgtt 108 O tatgtaagca gacagttitta ttgttcatga tigatatattt ttatc.ttgttg caatgta aca 114 O t cagagattt tdagacacaa cqtggctitt.c ccc.ggcc cat gaccaaaatc cct taacgtg 12 OO agttitt cqtt coactgagcg tdagaccc.cg tagaaaagat caaaggat.ct tcttgagat c 126 O

US 2009/006925.6 A1 Mar. 12, 2009 55

- Continued attgta acac ct cagt catt acacaggcct gtccaaagat gtc.ctittgaa ccaattic cca 378 O tacattattg taccc.cggct ggittatgcga ttctaaagtg taatgataat aagttcaatg 384 O gaac aggacc atgtacaaat gtcagcacaa tacaatgtac acatggaatt aagcc agtag 3900 tgtcaactica actgctgtta aatggcagtic tag cagalagg aggagaggta ataattagat 396 O ctgaaaatct cacagacaat gctaaaacca taatagtaca gct caaggaa cctdtagaaa 4 O2O t caattgtac aagacic caac aacaatacaa gaaaaagtat acatatggga C caggagcag 4 O8O cattt tatgc aagaggagaa gtaataggag atatalagaca agcacattgc aac attagta 414 O gaggaagatg gaatgacact ttaaaacaga tagctaaaaa attaa.gagaa caatttaata 42OO aaacaataag cct taaccaa toc to aggag gigg acctaga aattgtaatg cacacttitta 426 O attgtggagg ggaattitt to tactgtaata caacacagot gtttaatagt acttggaatg 432O agaatgatac tacctggaat aatacagoag gigt caaataa caatgaaact at cacacticc 438 O

Catgtagaat aaaacaaatt at aaa.caggt ggcaggaagt aggaaaag.ca atgitatgcc C 4 44 O citcc catcag toggaccaatt aattgtttat caaatat cac agggct atta ttaacaagag 4500 atggtggtga caacaataat acaatagaga cct tcagacic tigaggagga gatatgaggg 456 O acaattggag aagtgaatta tataaatata aagtagtaag aattgagcca ttaggaatag 462O Cacccaccala ggcaaagaga agagtggtgc aaa.gaga aaa aagagcagtg ggaataggag 468O Ctatgttcct tdggttcttg ggagcago ag gaagc act at ggg.cgcagog to agtgacgc 474. O tgacgg taca ggc.ca.gacta t tattgtctg gtatagtgca acagcaaaac aatttgctga 48OO gagdtatcga ggcgcaa.cag Catctgttgc aact cacagt Ctggggcatc aag cagotcC 486 O aggctagagt cctggctatg gaaagatacc taaaggat.ca acagctic ct a gggatttggg 492 O gttgct Ctgg aaaact catt to accacta atgtgccttg gaatgctagt tagtaata 498O aatctotgga caagatttgg catalacatga Cctggatgga gtgggacaga gaaattgaca 5040 attacacaaa attaatatac accittaattig aag catcgca gatccagcag gaaaagaatg 51OO aacaagaatt attggaattig gat agttggg caagtttgttg gagttggttt gaCatct caa 516 O aatggctgtg gtatatagga gtatt cataa tagtaatagg aggtttagta ggtttaaaaa 522 O tagtttittgc tigtactittct at agtaaata gagittaggca gggatactica ccattat cat 528 O ttcagacccd cct Cocagcc cc.gcggggac ccgacaggcc caaggaat c gaagaaggag 534 O gtggagagag agacagagac agat.ccgatc aattagtgac tigattctta gcact catct 54 OO gggacgatct gcggagcctg. tcct Cttica gctaccaccg Cttgagagaci ttact cttga 546 O ttgtagcgag gattgttggala Cttctgggac gcagggggtg ggaagcc ctgaagtattggit 552O ggaatcto ct gcaat attgg attcaggaac taaagaatag tectgttagt ttgcttalacg 558 O ccacagct at agcagtagcc gaggggacag at aggattat agaagtagta Caaaggattg 564 O gtagagct at t ct coacata cct agaagaa taaga caggg Cttagaaagg gctttgctat st OO aatagggcgc gcc.gagct cq Ctgat cagcc ticgactgtgc Cttct agttg C cagc Catct 576. O gttgtttgcc cct coccc.gt gcc titccttg accctggaag gtgcc acticc cactgtc.ctt 582O t cctaataaa atgaggaaat tcatcgc at ttctgagta ggtgt catt C tattotgggg 588 O ggtggggtgg gCaggacag Caagggggag gattgggaag acaat agcag gCatgctggg 594 O gaattit 5946 US 2009/006925.6 A1 Mar. 12, 2009 56

- Continued

<210 SEQ ID NO 11 <211 LENGTH: 54 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OO SEQUENCE: 11 atggattgga cittggat.ctt atttittagtt gctgctgcta citagagttca ttct 54

<210 SEQ ID NO 12 <211 LENGTH: 489 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OO SEQUENCE: 12 atgcggattt coaaac ct catct caggit co atttic catcc agtgctacct c to tctic ct c 6 O citcaactic cc attittct cac gigaagctggc att catgtct t cattgtcgg ctgtttcticc 12 O gcggggct Co. Ctaaaacgga agccaactgg gtgaatgtca ttt CCatct caaaaaaatt 18O gaagat ct cattcaatcc at gcatattgat gcgacgct ct atacggaatc cqatgtccac 24 O ccct cotgca aagtcaccgc gatgaagtgc tittct cotcg agctccaagt cattt Cocto 3OO gagt ccgggg atgcgt.cc at t catgatacg gtcgaaaatc tdatcatCct cqcgaacaac 360 t ccct ct cct C caatgggaa tt cacggaa ticcgggtgca aagaatgtga ggaactggag 42O gaaaaaaata ttaaagaatt tot coagt cc tttgtccata ttgtc.caaat gttcatcaac 48O acgt.cc tag 489

<210 SEQ ID NO 13 <211 LENGTH: 399 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OO SEQUENCE: 13 atggattgga cittggat.ctt atttittagtt gctgctgcta citagagttca ttctaactgg 6 O gtgaatgtaa taagtgattt gaaaaaaatt gaagat citta ttcaatctat gcatattgat 12 O gctactitt at atacggaaag tatgttcac cccagttgca aagta acagc aatgaagtgc 18O tittct cittgg agttacaagt tattt cactt gag to cqgag atgcaagtat t catgataca 24 O gtagaaaatc tdatcatcct agcaaacaac agtttgtc.tt ctaatgggaa totaacagaa 3OO tctggatgca aagaatgtgaggaactggag gaaaaaaata ttaaagaatt tttgcagagt 360 tttgtacata ttgtccaaat gttcatcaac acttcttga 399

<210 SEQ ID NO 14 <211 LENGTH: 399 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct US 2009/006925.6 A1 Mar. 12, 2009 57

- Continued <4 OO SEQUENCE: 14 atggattgga C9tggat.cct Ctttct citc gcggcggcga cqcgggtc.ca ttcCaactgg 6 O gtgaatgtca ttt cogatct caaaaaaatt gaagat citca ttcaatic cat gcatattgat 12 O gcgacgct ct atacggaatc catgtc.cac ccctic ctgca aagtic accgc gatgaagtgc 18O tittct c ct cq agcticcaagt cattt coct c gag to cqggg atgcqtc cat t catgatacg 24 O gtcgaaaatc tdatcatcct cqcgaacaac tocct ct cot coaatgggaa tdt cacggaa 3OO tccgggtgca aagaatgtgaggaactggag gaaaaaaata ttaaagaatt tdt CC agt cc 360 tttgtc. cata ttgtccaaat gttcatcaac acgt.cctag 399

<210 SEQ ID NO 15 <211 LENGTH: 399 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OO SEQUENCE: 15 atggactgga cctggat.cct gttcCtggtg gcc.gc.cgc.ca ccc.gc.gtgca CtcCaactgg 6 O gtgaacgtga t cagcgacct gaagaagatc gaggacctga t ccagagcat gca catcgac 12 O gccaccCtgt acaccgagag cacgtgcac cccagctgca aggtgaccgc catgaagtgc 18O titcCtgctgg agctgcaggt gat cagoctg gagagcggcg acgc.ca.gcat C cacgacacc 24 O gtggagaacc tdatcatCct ggccaacaac agcctgagca gcaacggcaa C9tgaccgag 3OO agcggctgca aggagtgcga ggagctggag gagaagaaca toaaggagtt CCtgcagagc 360 titcgtgcaca togtgcagat gttcatcaac accagotag 399

<210 SEQ ID NO 16 <211 LENGTH: 399 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OO SEQUENCE: 16 atggattgga cct ggat.cct ctittcttgtc gcc.gctgcca citcgagtaca ttcaaactgg 6 O gtaaatgtga titt cogacct taaaaaaatt gaagaccitta t coaaag cat gcacatagac 12 O gccacc ctitt at actgaatc cqacgtacac ccctic ct gca aagttaccgc catgaaatgt 18O tittct c ct cq aacticcaagt aattagcct c gaatc.cggag acgcct citat coacgacaca 24 O gttgaaaacc ticataatcct togcaaataac tot cittagot caaacggaaa tdt tactgaa 3OO tctggttgta aagaatgcga agaacttgaa gaaaaaaata taaaagaatt totgcaatca 360 tttgtc. caca togttcaaat gtttatcaat acct cittag 399

<210 SEQ ID NO 17 <211 LENGTH: 489 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic Construct

<4 OO SEQUENCE: 17

US 2009/006925.6 A1 Mar. 12, 2009 63

- Continued gcc.gcatcga tigatat coat togcatacgitt gtatictatat cataatatgt acatttatat 234 O tggct catgt ccaatatgac cqc catgttg acattgatta ttgac tagtt attaatagta 24 OO atcaattacg giggit cattag tt catagocc atatatggag titcc.gcgitta cataacttac 246 O ggtaaatggc cc.gc.ctggct gaccgc.ccala Caccc.ccgc C cattgacgt caataatgac 252O gtatgttcCC at agtaacgc caatagggac titt CC attga C9tcaatggg toggagtattt 2580 acgg taaact gcc cacttgg cagta catca agtgitat cat atgccaagtic cqc coccitat 264 O tgacgt caat gacggtaaat ggc.ccgc.ctg gcattatgcc cagtacatga Ccttacggga 27 OO ctitt cotact toggcagtaca totacg tatt agt catcgct attaccatgg tdatgcggitt 276 O ttggcagtac at Caatgggc gtggatagcg gtttgactica C9gggattt C Caagttct coa 282O c cc cattgac git caatggga gtttgttittg gcaccaaaat caacgggact titccaaaatg 288O tcgtaacaac tocccc.cat tacgcaaat gggcgg tagg catgitacggit gggaggit ct a 294 O tataag caga gct cqtttag talaccgt.ca gat.cgc.ctgg agacgc.cat C Cacgctgttt 3 OOO tgacct coat agaaga cacc gggaccgatc cagcct CCC gggcgc.gcgt. Caccaccat 3 O 6 O ggattggacc tigatcct ct ttcttgtc.gc cqctgcc act cagtacatt caaactgggit 312 O aaatgtgatt toccaccitta aaaaaattga agacctitatic caaag catgc acatagacgc 318O cacc ctittat actgaatc.cg acgtacaccc ctic ct gcaaa gttaccgc.ca tdaaatgttt 324 O totcct cqaa citccaagtaa ttagcctcqa atc.cggagac goctotatoc acgacacagt 33 OO tgaaaacctic ataatccttg caaataactic ticttagctica aacggaaatgttactgaatc 3360 tggttgtaaa gaatgcgaag aacttgaaga aaaaaatata aaagaattitc togcaat catt 342O tgtc.ca catc gttcaaatgt titat caatac ct cittagtgagtc.gacgggc gacgcgaaac 3480 ttgggcc.cac tdgagaggcg cgc.cgagctic gctgat Cagc Ctcgactgtg cct tctagtt 354 O gccago catc tdttgtttgc ccctic ccc.cg togcct tcctt gaccctggaa gotgccactic 36OO c cactgtc.ct titcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 366 O

Ct attctggg giggtggggtggggcaggaca gcaa.ggggga gattgggala gacalata.gca 372 O ggcatgctgg ggaattit 3737

1-397. (canceled) 402. The modified polynucleotide of claim 398, addition 398. A modified polynucleotide comprising: a nucleic acid ally comprising a non-native leader sequence. sequence comprising one or more Surrogate codons in place of a corresponding naturally-occurring codon having adenine 403. The modified polynucleotide of claim 398, addition (A), thymine (T), or uracil (U) in the wobble position; ally comprising a human non-native leader sequence. wherein the Surrogate codon encodes the same amino acid as 404. The modified polynucleotide of claim 398, addition the naturally-occurring codon. ally comprising an immunoglobulin leader sequence. 399. The modified polynucleotide of claim 398, wherein 405. The modified polynucleotide of claim 398, addition the Surrogate codons encode any of the amino acids alanine, ally comprising (a) an IgE leader sequence or (b) a leader arginine, leucine, proline, glutamic acid, glycine, isoleucine, sequence that hybridizes to an IgE leader sequence under serine, threonine, or valine. stringent conditions. 400. The modified polynucleotide of claim 399, wherein the Surrogate codons comprise cytosine (C) or guanine (G) at 406. The modified polynucleotide of claim 398, addition the wobble position. ally comprising a leader sequence comprising SEQID NO: 401. The modified polynucleotide of claim 399, wherein 11. the Surrogate codon encoding alanine is GCG, encoding argi 407. The modified polynucleotide of claim 406, wherein nine is CGG or AGG, encoding leucine is CTC, encoding the leader sequence has at least 90% sequence identity to the proline is CCT or CCG, encoding glutamic acid is GAG, nucleic acid sequence of SEQID NO: 11. encoding glycine is GGG, encoding isoleucine is ATT, encod 408. The modified polynucleotide of claim 406, wherein ing serine is TCC, encoding threonine is ACG, and encoding the leader sequence has at least 95% sequence identity to the valine is GTC. nucleic acid sequence of SEQID NO: 11. US 2009/006925.6 A1 Mar. 12, 2009 64

409. The modified polynucleotide of claim 406, wherein 422. The modified polynucleotide of claim 398, wherein the leader sequence is the nucleic acid sequence of SEQ ID the modified polynucleotide sequence has at least 90% NO: 11. sequence identity to the nucleic acid sequence of any of SEQ 410. The modified polynucleotide of claim 398, wherein ID NOS: 12-16. the modified polynucleotide encodes a viral, bacterial, pro 423. The modified polynucleotide of claim 398, wherein tist, fungal, plant, or animal polypeptide. the modified polynucleotide sequence has at least 95% 411. The modified polynucleotide of claim 410, wherein sequence identity to the nucleic acid sequence of any of SEQ the modified polynucleotide encodes a mammalian polypep ID NOS: 12-16. tide. 424. A composition comprising the modified polynucle 412. The modified polynucleotide of claim 410. wherein otide of claim 398 and a pharmaceutically acceptable vector. the viral polypeptide is an HPV 16 polypeptide or an HIV-1 425. A composition comprising the nucleic acid sequence of any of SEQID NOS: 1, 3, 5, 12, 13, 14, 15, or 16. polypeptide. 426. A method for preparing a polynucleotide that provides 413. The modified polynucleotide of claim 398, wherein enhanced expression of a gene comprising: the modified polynucleotide comprises the open reading assembling oligonucleotides comprising Surrogate codons frame (ORF) for the HPV 16 E7 gene, HIV-1 gag gene, or to form a modified polynucleotide comprising one or gp160 envelope gene. more Surrogate codons in place of a corresponding natu 414. The modified polynucleotide of claim 398, wherein rally-occurring codon having adenine (A), thymine (T), the Surrogate codons are a randomized selection of at least or uracil (U) in the wobble position; wherein the surro about 10% of the codons in said modified polynucleotide that gate codon encodes the same amino acid as the natu encode for any of the amino acids alanine, arginine, leucine, rally-occurring codon. proline, glutamic acid, glycine, isoleucine, serine, threonine 427. The method of claim 426, wherein the surrogate and valine. codon encodes any of the amino acids alanine, arginine, leu 415. The modified polynucleotide of claim 398, wherein cine, proline, glutamic acid, glycine, isoleucine, serine, threo the Surrogate codons are a randomized selection of at least nine and valine. about 50% of the codons in said modified polynucleotide that 428. The method of claim 426, wherein the surrogate encode for any of the amino acids alanine, arginine, leucine, codons comprises cytosine (C) or guanine (G) at the wobble proline, glycine, isoleucine, serine, threonine and Valine. position. 416. The modified polynucleotide of claim 398, wherein 429. The method of claim 426, wherein the surrogate the Surrogate codons are a randomized selection of at least codon encodingalanine is GCG, encoding arginine is CGG or about 90% of the codons in said modified polynucleotide that AGG, encoding leucine is CTC, encoding proline is CCT or encode for any of the amino acids alanine, arginine, leucine, CCG, encoding glutamic acid is GAG, encoding glycine is proline, glycine, isoleucine, serine, threonine and Valine. GGG, encoding iso is ATT, encoding serine is TCC, encoding 417. The modified polynucleotide of claim 398, wherein threonine is ACG, and encoding valine is GTC. the modified polynucleotide is a DNA molecule. 430. The method of claim 426, additionally comprising 418. The modified polynucleotide of claim 398, wherein adding a non-native leader sequence to the modified poly the modified polynucleotide is an RNA molecule. nucleotide. 419. The modified polynucleotide of claim 398, wherein 431. The method of claim 426, additionally comprising adding a human non-native leader sequence to the modified the nucleic acid sequence comprises any of polynucleotide. (a) the nucleic acid sequence encoding any of SEQ ID 432. The method of claim 426, additionally comprising NOS: 2,4, or 6: adding an immunoglobulin leader sequence to the modified (b) an immunogenic encoding portion of SEQID NOS: 2. polynucleotide. 4 or 6; or 433. The method of claim 432, wherein the immunoglobu (c) a nucleic acid sequence that hybridizes under stringent lin leader sequence is: (a) an IgE leader sequence or (b) a conditions to the nucleic acid sequence encoding any of leader sequence that hybridizes to an IgE leader sequence SEQID NOS: 24, or 6. under stringent conditions. 420. The modified polynucleotide of claim 398, wherein 434. The method of claim 433, wherein the immunoglobu the nucleic acid sequence comprises any of lin leader sequence is an IgE leader sequence. (a) a nucleic acid sequence having at least about 70% 435. The method of claim 432, additionally comprising sequence identity to the nucleic acid sequence of SEQ adding to the modified polynucleotide a leader sequence ID NO: 14; or comprising SEQID NO: 11. (b) a nucleic acid sequence that hybridizes to SEQID NO: 436. The method of claim 432, additionally comprising 14 under Stringent conditions. adding to the modified polynucleotide a leader sequence hav ing at least 95% sequence identity to the nucleic acid 421. The modified polynucleotide of claim 398, wherein sequence of SEQID NO: 11. the nucleic acid sequence comprises any of 437. A method for preparing a modified polynucleotide (a) the nucleic acid sequence encoding any of SEQ ID that provides enhanced expression of a polynucleotide NOS: 12-16: sequence comprising: (b) an immunogenic encoding portion of SEQ ID NOS: providing a polynucleotide sequence having a plurality of 12-16; or codons having the nucleotides adenine (A) or uracil (U) (c) a nucleic acid sequence that hybridizes under stringent or thymine (T) at the wobble position; substituting one conditions to the nucleic acid sequence encoding any of or more codons having the nucleotides adenine (A) or SEQID NOS: 12-16. uracil (U) or thymine (T) at the wobble position with a US 2009/006925.6 A1 Mar. 12, 2009 65

surrogate codon having the nucleotides cytosine (C) or position; wherein the surrogate codon encodes the same guanine (G) at the wobble position; wherein the surro- amino acid as the naturally-occurring codon. gate codon encodes the same amino acid as the codons 439. A method of preventing or treating a disease in a having the nucleotides adenine (A) or uracil (U) or mammal comprising: administering to the mammal an effec thymine (T) at the wobble position; and attaching a tive amount of a composition comprising a nucleic acid leader sequence to the polynucleotide sequence, sequence comprising one or more surrogate codons in place wherein the leader sequence is a non-native leader of a corresponding naturally-occurring codon having adenine sequence to the polynucleotide sequence. (A), thymine (T), or uracil (U) in the wobble position; 438. A method for enhancing expression of a gene com- wherein the surrogate codon encodes the same amino acid as prising: the naturally-occurring codon. expressing in vivo or in vitro the modified polynucleotide 440. The method of claim 439, wherein the composition is modified polynucleotide comprising: a nucleic acid administered parenterally, mucosally, subcutaneously, or sequence comprising one or more surrogate codons in intramuscularly. place of a corresponding naturally-occurring codon hav ing adenine (A), thymine (T), or uracil (U) in the wobble ck c. c. c :