Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof

Abstract

The present invention relates to polynucleotides encoding immunogenic HIV polypeptides. Uses of the polynucleotides in applications including immunization, generation of packaging cell lines, and production of HIV polypeptides are also described. Polynucleotides encoding antigenic HIV polypeptides are described, as are uses of these polynucleotides and polypeptide products therefrom, including formulations of immunogenic compositions and uses thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional Patent Application Ser. Nos. 60/349,728, filed 16 Jan. 2002, and 60/316,860, filed 31 Aug. 2001, from which priority is claimed under 35 USC .sctn. 119(e)(1), and which applications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

[0002] Polynucleotides encoding antigenic HIV polypeptides (e.g., those shown in Table C) are described, as are uses of these polynucleotides and polypeptide products including formulations of immunogenic compositions and uses thereof.

BACKGROUND OF THE INVENTION

[0003] Acquired immune deficiency syndrome (AIDS) is recognized as one of the greatest health threats facing modern medicine. There is, as yet, no cure for this disease.

[0004] In 1983-1984, three groups independently identified the suspected etiological agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983) Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses (Gallo, Essex & Gross, eds., 1984); Vilmer et al. (1984) The Lancet 1:753; Popovic et al. (1984) Science 224:497-500; Levy et al. (1984) Science 225:840-842. These isolates were variously called lymphadenopathy-associated virus (LAV), human T-cell lymphotropic virus type III (HTLV-III), or AIDS-associated retrovirus (ARV). All of these isolates are strains of the same virus, and were later collectively named Human Immunodeficiency Virus (HIV). With the isolation of a related AIDS-causing virus, the strains originally called HIV are now termed HIV-1 and the related virus is called HIV-2 See, e.g., Guyader et al. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) Science 233:343-346; Clavel et al. (1986) Nature 324:691-695.

[0005] A great deal of information has been gathered about the HIV virus, however, to date an effective vaccine has not been identified. Several targets for vaccine development have been examined including the env and Gag gene products encoded by HIV. Gag gene products include, but are not limited to, Gag-polymerase and Gag-protease. Env gene products include, but are not limited to, monomeric gp120 polypeptides, oligomeric gp140 polypeptides and gp160 polypeptides.

[0006] Haas, et al., (Current Biology 6(3):315-324, 1996) suggested that selective codon usage by HIV-1 appeared to account for a substantial fraction of the inefficiency of viral protein synthesis. Andre, et al., (J. Virol. 72(2): 1497-1503, 1998) described an increased immune response elicited by DNA vaccination employing a synthetic gp120 sequence with modified codon usage. Schneider, et al., (J. Virol. 71(7):4892-4903, 1997) discuss inactivation of inhibitory (or instability) elements (INS) located within the coding sequences of the Gag and Gag-protease coding sequences.

[0007] The Gag proteins of HIV-1 are necessary for the assembly of virus-like particles. HIV-1 Gag proteins are involved in many stages of the life cycle of the virus including, assembly, virion maturation after particle release, and early post-entry steps in virus replication. The roles of HIV-1 Gag proteins are numerous and complex (Freed, E. O., Virology 251:1-15, 1998).

[0008] Wolf, et al., (PCT International Application, WO 96/30523, published 3 Oct. 1996; European Patent Application, Publication No. 0 449 116 A1, published 2 Oct. 1991) have described the use of altered pr55 Gag of HIV-1 to act as a non-infectious retroviral-like particulate carrier, in particular, for the presentation of immunologically important epitopes. Wang, et al., (Virology 200:524-534, 1994) describe a system to study assembly of HIV Gag-.beta.-galactosidase fusion proteins into virions. They describe the construction of sequences encoding HIV Gag-.beta.-galactosidase fusion proteins, the expression of such sequences in the presence of HIV Gag proteins, and assembly of these proteins into virus particles.

[0009] Shiver, et al., (PCT International Application, WO 98/34640, published 13 Aug. 1998) described altering HIV-1 (CAM1) Gag coding sequences to produce synthetic DNA molecules encoding HIV Gag and modifications of HIV Gag. The codons of the synthetic molecules were codons preferred by a projected host cell.

[0010] Recently, use of HIV Env polypeptides in immunogenic compositions has been described. (see, U.S. Pat. No. 5,846,546 to Hurwitz et al., issued Dec. 8, 1998, describing immunogenic compositions comprising a mixture of at least four different recombinant virus that each express a different HIV env variant; and U.S. Pat. No. 5,840,313 to Vahlne et al., issued Nov. 24, 1998, describing peptides which correspond to epitopes of the HIV-1 gp120 protein). In addition, U.S. Pat. No. 5,876,731 to Sia et al, issued Mar. 2, 1999 describes candidate vaccines against HIV comprising an amino acid sequence of a T-cell epitope of Gag linked directly to an amino acid sequence of a B-cell epitope of the V3 loop protein of an HIV-1 isolate containing the sequence GPGR.

SUMMARY OF THE INVENTION

[0011] Described herein are novel HIV sequences, polypeptides encoded by these novel sequences, and synthetic expression cassettes generated from these and other HIV sequences. In one aspect, the present invention relates to improved HIV expression cassettes. In a second aspect, the present invention relates to generating an immune response in a subject using the expression cassettes of the present invention. In a further aspect, the present invention relates to generating an immune response in a subject using the expression cassettes of the present invention, as well as, polypeptides encoded by the expression cassettes of the present invention. In another aspect, the present invention relates to enhanced vaccine technologies for the induction of potent neutralizing antibodies and/or cellular immune responses against HIV in a subject.

[0012] In certain embodiments, the present invention relates to synthetic polynucleotides and/or expression cassettes encoding HIV polypeptides, including, but not limited to, Env, Gag, Pol, RT, Int, Prot, Vpr, Vpu, Vif, Nef, Tat, Rev and/or fragments or combinations thereof. In addition, the present invention also relates to improved expression of HIV polypeptides and production of virus-like particles. Synthetic expression cassettes encoding the HIV polypeptides (e.g., Gag-, pol-, protease (prot)-, reverse transcriptase, integrase, RNAseH, Tat, Rev, Nef, Vpr, Vpu, Vif and/or Env-containing polypeptides) are described, as are uses of the expression cassettes. Mutations in some of the genes are described that reduce or eliminate the activity of the gene product without adversely affecting the ability of the gene product to generate an immune response. Exemplary synthetic polynucleotides include, but are not limited to, GagComplPolmut.SF2 (SEQ ID NO:9), GagComplPolmutAtt.SF2 (SEQ ID NO:10), GagComplPolmutIna.SF2 (SEQ ID NO:11), gagCpolInaTatRevNef.opt_B (SEQ ID NO:12), GagPolmutAtt.SF2 (SEQ ID NO: 13), GagPolmutIna.SF2 (SEQ ID NO: 14), GagProtInaRTmut.SF2 (SEQ ID NO: 15), GagProtInaRTmutTatRevNef.opt_B (SEQ ID NO:16), GagRTmut.SF2 (SEQ ID NO:17), GagTatRevNef.opt_B (SEQ ID NO:18), gp140.modSF162.CwtLmod (SEQ ID NO:19), gp140.modSF162.CwtLnat (SEQ ID NO:20), gp160.modSF162.delV2.mut7 (SEQ ID NO:21), gp160.modSF162.delV2.mut8 (SEQ ID NO:22), int.opt.mut.SF2 (SEQ ID NO:23), int.opt.SF2 (SEQ ID NO:24), nef.D125G.-myr.opt.SF162 (SEQ ID NO:25), nef.D107G.-myr18.opt.SF162 (SEQ ID NO:26), nef.opt.D125G.SF162 (SEQ ID NO:27), nef.opt.SF162 (SEQ ID NO:28), p15RnaseH.opt.SF2 (SEQ ID NO:29), p2Pol.opt.YMWM.SF2 (SEQ ID NO:30), p2PolInaopt.YM.SF2 (SEQ ID NO:31), p2Polopt.SF2 (SEQ ID NO:32), p2PolTatRevNef.opt.native_B (SEQ ID NO:33), p2PolTatRevNef.opt_B (SEQ ID NO:34), pol.opt.SF2 (SEQ ID NO:35), prot.opt.SF2 (SEQ ID NO:36), protIna.opt.SF2 (SEQ ID NO:37), protInaRT.YM.opt.SF2 (SEQ ID NO:38), protInaRT.YMWM.opt.SF2 (SEQ ID NO:39), ProtInaRTmut.SF2 (SEQ ID NO:40), protRT.opt.SF2 (SEQ ID NO:41), ProtRT.TatRevNef.opt_B (SEQ ID NO:42), ProtRTTatRevNef.opt_B (SEQ ID NO:43), rev.exon1.sub.--2.M5-10.opt.SF162 (SEQ ID NO:44), rev.exon1.sub.--2.opt.SF162 (SEQ ID NO:45), RT.opt.SF2 (mutant) (SEQ ID NO:46), RT.opt.SF2 (native) (SEQ ID NO:47), RTmut.SF2 (SEQ ID NO:48), tat.exon1.sub.--2.opt.C22-37.SF2 (SEQ ID NO:49), tat.exon1.sub.--2.opt.C37.SF2 (SEQ ID NO:50), TatRevNef.opt.native.SF162 (SEQ ID NO:51), TatRevNef.opt.SF162 (SEQ ID NO:52), TatRevNefGag B (SEQ ID NO:53), TatRevNefgagCpolIna B (SEQ ID NO:54), TatRevNefGagProtInaRTmut B (SEQ ID NO:55), TatRevNefp2Pol.opt_B, (SEQ ID NO:56) TatRevNefprotRTopt B (SEQ ID NO:57), vif.opt.SF2 (SEQ ID NO:58), vpr.opt.SF2 (SEQ ID NO:59), and vpu.opt.SF162 (SEQ ID NO:60).

[0013] Thus, one aspect of the present invention relates to expression cassettes and polynucleotides contained therein. The expression cassettes typically include an HIV-polypeptide encoding sequence inserted into an expression vector backbone. In one embodiment, an expression cassette comprises a polynucleotide sequence encoding one or more polypeptides, wherein the polynucleotide sequence comprises a sequence having between about 85% to 100% and any integer values therebetween, for example, at least about 85%, preferably about 90%, more preferably about 95%, and more preferably about 98% sequence identity to the sequences taught in the present specification.

[0014] The polynucleotides encoding the HIV polypeptides of the present invention may also include sequences encoding additional polypeptides. Such additional polynucleotides encoding polypeptides may include, for example, coding sequences for other viral proteins (e.g., hepatitis B or C or other HIV proteins, such as, polynucleotide sequences encoding an HIV Gag polypeptide, polynucleotide sequences encoding an HIV Env polypeptide and/or polynucleotides encoding one or more of vif, vpr, tat, rev, vpu and nef); cytokines or other transgenes.

[0015] In one embodiment, the sequence encoding the HIV Pol polypeptide(s) can be modified by deletions of coding regions corresponding to reverse transcriptase and integrase. Such deletions in the polymerase polypeptide can also be made such that the polynucleotide sequence preserves T-helper cell and CTL epitopes. Other antigens of interest may be inserted into the polymerase as well.

[0016] In another embodiment, an expression cassette comprises a polynucleotide sequence encoding a polypeptide, for example, GagComplPolmut.SF2 (SEQ ID NO:9), GagComplPolmutAtt.SF2 (SEQ ID NO:10), GagComplPolmutIna.SF2 (SEQ ID NO:11), gagCpolInaTatRevNef.opt_B (SEQ ID NO:12), GagPolmutAtt.SF2 (SEQ ID NO:13), GagPolmutIna.SF2 (SEQ ID NO:14), GagProtInaRTmut.SF2 (SEQ ID NO:15), GagProtInaRTmutTatRevNef.opt_B (SEQ ID NO:16), GagRTmut.SF2, (SEQ ID NO:17) GagTatRevNef.opt_B (SEQ ID NO:18), gp140.modSF162.CwtLmod (SEQ ID NO:19), gp140.modSF162.CwtLnat (SEQ ID NO:20), gp160.modSF162.delV2.mut7 (SEQ ID NO:21), gp160.modSF162.delV2.mut8 (SEQ ID NO:22), int.opt.mut.SF2 (SEQ ID NO:23), int.opt.SF2 (SEQ ID NO:24), nef.D125G.-myr.opt.SF162 (SEQ ID NO:25), nef.D107G.-myr18.opt.SF162 (SEQ ID NO:26), nef.opt.D125G.SF162 (SEQ ID NO:27), nef.opt.SF162 (SEQ ID NO:28), p15RnaseH.opt.SF2 (SEQ ID NO:29), p2Pol.opt.YMWM.SF2 (SEQ ID NO:30), p2PolInaopt.YM.SF2, (SEQ ID NO:31) p2Polopt.SF2 (SEQ ID NO:32), p2PolTatRevNef.opt.native_B (SEQ ID NO:33), p2PolTatRevNef.opt_B (SEQ ID NO:34), pol.opt.SF2 (SEQ ID NO:35), prot.opt.SF2 (SEQ ID NO:36), protIna.opt.SF2 (SEQ ID NO:37), protInaRT.YM.opt.SF2 (SEQ ID NO:38), protInaRT.YMWM.opt.SF2 (SEQ ID NO:39), ProtInaRTmut.SF2 (SEQ ID NO:40), protRT.opt.SF2 (SEQ ID NO:41), ProtRT.TatRevNef.opt_B (SEQ ID NO:42), ProtRTTatRevNef.opt_B (SEQ ID NO:43), rev.exon1.sub.--2.M5-10.opt.SF162 (SEQ ID NO:44), rev.exon1.sub.--2.opt.SF162 (SEQ ID NO:45), RT.opt.SF2 (mutant) (SEQ ID NO:46), RT.opt.SF2 (native) (SEQ ID NO:47), RTmut.SF2 (SEQ ID NO:48), tat.exon1.sub.--2.opt.C22-37.SF2 (SEQ ID NO:49), tat.exon1.sub.--2.opt.C37.SF2 (SEQ ID NO:50), TatRevNef.opt.native.SF162 (SEQ ID NO:51), TatRevNef.opt.SF162 (SEQ ID NO:52), TatRevNefGag B (SEQ ID NO:53), TatRevNefgagCpolIna B (SEQ ID NO:54), TatRevNefGagProtInaRTmut B (SEQ ID NO:55), TatRevNefp2Pol.opt_B (SEQ ID NO:56), TatRevNefprotRTopt B (SEQ ID NO:57), vif.opt.SF2 (SEQ ID NO:58), vpr.opt.SF2 (SEQ ID NO:59), and vpu.opt.SF162 (SEQ ID NO:60), wherein the polynucleotide sequence encoding the polypeptide comprises a sequence having between about 85% to 100% and any integer values therebetween, for example, at least about 85%, preferably about 90%, more preferably about 95%, and most preferably about 98% sequence identity to the sequences taught in the present specification.

[0017] The native and synthetic polynucleotide sequences encoding the HIV polypeptides of the present invention typically have between about 85% to 100% and any integer values therebetween, for example, at least about 85%, preferably about 90%, more preferably about 95%, and more preferably about 98% sequence identity to the sequences taught herein. Further, in certain embodiments, the polynucleotide sequences encoding the HIV polypeptides of the invention will exhibit 100% sequence identity to the sequences taught herein.

[0018] The polynucleotides of the present invention can be produced by recombinant techniques, synthetic techniques, or combinations thereof.

[0019] The present invention further includes recombinant expression systems for use in selected host cells, wherein the recombinant expression systems employ one or more of the polynucleotides and expression cassettes of the present invention. In such systems, the polynucleotide sequences are operably linked to control elements compatible with expression in the selected host cell. Numerous expression control elements are known to those in the art, including, but not limited to, the following: transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences. Exemplary transcription promoters include, but are not limited to those derived from CMV, CMV+intron A, SV40, RSV, HIV-Ltr, MMLV-ltr, and metallothionein.

[0020] In another aspect the invention includes cells comprising one or more of the expression cassettes of the present invention where the polynucleotide sequences are operably linked to control elements compatible with expression in the selected cell. In one embodiment such cells are mammalian cells. Exemplary mammalian cells include, but are not limited to, BHK, VERO, HT1080, 293, RD, COS-7, and CHO cells. Other cells, cell types, tissue types, etc., that may be useful in the practice of the present invention include, but are not limited to, those obtained from the following: insects (e.g., Trichoplusia ni (Tn5) and Sf9), bacteria, yeast, plants, antigen presenting cells (e.g., macrophage, monocytes, dendritic cells, B-cells, T-cells, stem cells, and progenitor cells thereof), primary cells, immortalized cells, tumor-derived cells.

[0021] In a further aspect, the present invention includes compositions for generating an immunological response, where the composition typically comprises at least one of the expression cassettes of the present invention and may, for example, contain combinations of expression cassettes such as one or more expression cassettes carrying a Pol-derived-polypeptide-encoding polynucleotide, one or more expression cassettes carrying a Gag-derived-polypeptide-encoding polynucleotide, one or more expression cassettes carrying accessory polypeptide-encoding polynucleotides (e.g., native or synthetic vpu, vpr, nef, vif, tat, rev), and/or one or more expression cassettes carrying an Env-derived-polypeptide-encoding polynucleotide. Such compositions may further contain an adjuvant or adjuvants. The compositions may also contain one or more HIV polypeptides. The HIV polypeptides may correspond to the polypeptides encoded by the expression cassette(s) in the composition, or may be different from those encoded by the expression cassettes. In compositions containing both expression cassettes (or polynucleotides of the present invention) and polypeptides, various expression cassettes of the present invention can be mixed and/or matched with various HIV polypeptides described herein.

[0022] In another aspect the present invention includes methods of immunization of a subject. In the method any of the above described compositions are into the subject under conditions that are compatible with expression of the expression cassette(s) in the subject. In one embodiment, the expression cassettes (or polynucleotides of the present invention) can be introduced using a gene delivery vector. The gene delivery vector can, for example, be a non-viral vector or a viral vector. Exemplary viral vectors include, but are not limited to eucaryotic layered vector initiation systems, Sindbis-virus (or other alphavirus) derived vectors, retroviral vectors, and lentiviral vectors. Other exemplary vectors include, but are not limited to, pCMVKm2, pCMV6a, pCMV-link, and pCMVPLEdhfr. Compositions useful for generating an immunological response can also be delivered using a particulate carrier (e.g., PLG or CTAB-PLG microparticles). Further, such compositions can be coated on, for example, gold or tungsten particles and the coated particles delivered to the subject using, for example, a gene gun. The compositions can also be formulated as liposomes. In one embodiment of this method, the subject is a mammal and can, for example, be a human.

[0023] In a further aspect, the invention includes methods of generating an immune response in a subject. Any of the expression cassettes described herein can be expressed in a suitable cell to provide for the expression of the HIV polypeptides encoded by the polynucleotides of the present invention. The polypeptide(s) are then isolated (e.g., substantially purified) and administered to the subject in an amount sufficient to elicit an immune response. In certain embodiments, the methods comprise administration of one or more of the expression cassettes or polynucleotides of the present invention, using any of the gene delivery techniques described herein. In other embodiments, the methods comprise co-administration of one or more of the expression cassettes or polynucleotides of the present invention and one or more polypeptides, wherein the polypeptides can be expressed from these polynucleotides or can be other HIV polypeptides. In other embodiments, the methods comprise co-administration of multiple expression cassettes or polynucleotides of the present invention. In still further embodiments, the methods comprise co-administration of multiple polypeptides, for example polypeptides expressed from the polynucleotides of the present invention and/or other HIV polypeptides.

[0024] The invention further includes methods of generating an immune response in a subject, where cells of a subject are transfected with any of the above-described expression cassettes or polynucleotides of the present invention, under conditions that permit the expression of a selected polynucleotide and production of a polypeptide of interest (e.g., encoded by any expression cassette of the present invention). By this method an immunological response to the polypeptide is elicited in the subject. Transfection of the cells may be performed ex vivo and the transfected cells are reintroduced into the subject. Alternately, or in addition, the cells may be transfected in vivo in the subject. The immune response may be humoral and/or cell-mediated (cellular). In a further embodiment, this method may also include administration of an HIV polypeptides before, concurrently with, and/or after introduction of the expression cassette into the subject.

[0025] The polynucleotides of the present invention may be employed singly or in combination. The polynucleotides of the present invention, encoding HIV-derived polypeptides, may be expressed in a variety of ways, including, but not limited to the following: a polynucleotide encoding a single gene product (or portion thereof) expressed from a promoter; multiple polynucleotides encoding a more than one gene product (or portion thereof) (e.g., polycistronic coding sequences); multiple polynucleotides in-frame to produce a single polyprotein; and, multiple polynucleotides in-frame to produce a single polyprotein wherein the polyprotein has protein cleavage sites between one or more of the polypeptides comprising the polyprotein.

[0026] These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

[0027] FIGS. 1A to 1D depict the nucleotide sequence of HIV Type C 8.sub.--5_TV1_C.ZA (SEQ ID NO:1; referred to herein as TV1). Various regions are shown in Table A.

[0028] FIGS. 2A-C depicts an alignment of Env polypeptides from various HIV isolates (SF162, SEQ ID NO:2; TV1.8.sub.--2, SEQ ID NO:3; TV1.8.sub.--5, SEQ ID NO:4; TV2.12-5/1, SEQ ID NO:5; Consensus Sequence, SEQ ID NO:6). The regions between the arrows indicate regions (of TV1 and TV2 clones, both HIV Type C isolates) in the beta and/or bridging sheet region(s) that can be deleted and/or truncated. The "*" denotes N-linked glycosylation sites (of TV1 and TV2 clones), one or more of which can be modified (e.g., deleted and/or mutated).

[0029] FIG. 3 presents a schematic diagram showing the relationships between the following forms of the HIV Env polypeptide: gp160, gp140, gp120, and gp41.

[0030] FIG. 4 presents exemplary data concerning transactivation activity of Tat mutants on LTR-CAT plasmid expression in 293 cells.

[0031] FIG. 5 presents exemplary data concerning export activity of Rev mutants monitored by CAT expression.

[0032] FIG. 6, sheets 1 and 2, presents the sequence of GagComplPolmut.SF2 (SEQ ID NO:9).

[0033] FIG. 7, sheets 1 and 2, presents the sequence of GagComplPolmutAtt.SF2 (SEQ ID NO:10).

[0034] FIG. 8, sheets 1 and 2, presents the sequence of GagComplPolmutIna.SF2 (SEQ ID NO:11).

[0035] FIG. 9, sheets 1 and 2, presents the sequence of gagCpolInaTatRevNef.opt_B (SEQ ID NO:12).

[0036] FIG. 10, sheets 1 and 2, presents the sequence of GagPolmutAtt.SF2 (SEQ ID NO:13).

[0037] FIG. 11, sheets 1 and 2, presents the sequence of GagPolmutIna.SF2 (SEQ ID NO:14).

[0038] FIG. 12, sheets 1 and 2, presents the sequence of GagProtInaRTmut.SF2 (SEQ ID NO:15).

[0039] FIG. 13, sheets 1 and 2, presents the sequence of GagProtInaRTmutTatRevNef.opt_B (SEQ ID NO:16).

[0040] FIG. 14, sheets 1 and 2, presents the sequence of GagRTmut.SF2 (SEQ ID NO:17).

[0041] FIG. 15, presents the sequence of GagTatRevNef.opt_B (SEQ ID NO:18).

[0042] FIG. 16, presents the sequence of gp140.modSF162.CwtLmod (SEQ ID NO:19).

[0043] FIG. 17, presents the sequence of gp140.modSF162.CwtLnat (SEQ ID NO:20).

[0044] FIG. 18, presents the sequence of gp160.modSF162.delV2.mut7 (SEQ ID NO:21).

[0045] FIG. 19, presents the sequence of gp160.modSF162.delV2.mut8 (SEQ ID NO:22).

[0046] FIG. 20, presents the sequence of int.opt.mut.SF2 (SEQ ID NO:23).

[0047] FIG. 21, presents the sequence of int.opt.SF2 (SEQ ID NO:24).

[0048] FIG. 22, presents the sequence of nef.D125G.-myr.opt.SF162 (SEQ ID NO:25).

[0049] FIG. 23, presents the sequence of nef.D107G.-myr18.opt.SF162 (SEQ ID NO:26).

[0050] FIG. 24, presents the sequence of nef.opt.D125G.SF162 (SEQ ID NO:27).

[0051] FIG. 25, presents the sequence of nef.opt.SF162 (SEQ ID NO:28).

[0052] FIG. 26, presents the sequence of p15RnaseH.opt.SF2 (SEQ ID NO:29).

[0053] FIG. 27, presents the sequence of p2Pol.opt.YMWM.SF2 (SEQ ID NO:30).

[0054] FIG. 28, presents the sequence of p2PolInaopt.YM.SF2 (SEQ ID NO:31).

[0055] FIG. 29, presents the sequence of p2Polopt.SF2 (SEQ ID NO:32).

[0056] FIG. 30, presents the sequence of p2PolTatRevNef.opt.native_B (SEQ ID NO:33).

[0057] FIG. 31, sheets 1 and 2, presents the sequence of p2PolTatRevNef.opt_B (SEQ ID NO:34).

[0058] FIG. 32, presents the sequence of pol.opt.SF2 (SEQ ID NO:35).

[0059] FIG. 33, presents the sequence of prot.opt.SF2 (SEQ ID NO:36).

[0060] FIG. 34, presents the sequence of protIna.opt.SF2 (SEQ ID NO:37).

[0061] FIG. 35, presents the sequence of protInaRT.YM.opt.SF2 (SEQ ID NO:38).

[0062] FIG. 36, presents the sequence of protInaRT.YMWM.opt.SF2 (SEQ ID NO:39).

[0063] FIG. 37, presents the sequence of ProtInaRTmut.SF2 (SEQ ID NO:40).

[0064] FIG. 38, presents the sequence of protRT.opt.SF2 (SEQ ID NO:41).

[0065] FIG. 39, presents the sequence of ProtRT.TatRevNef.opt_B (SEQ ID NO:42).

[0066] FIG. 40, presents the sequence of ProtRTTatRevNef.opt_B (SEQ ID NO:43).

[0067] FIG. 41, presents the sequence of rev.exon1.sub.--2.M5-10.opt.SF162 (SEQ ID NO:44).

[0068] FIG. 42, presents the sequence of rev.exon1.sub.--2.opt.SF162 (SEQ ID NO:45).

[0069] FIG. 43, presents the sequence of RT.opt.SF2 (mutant) (SEQ ID NO:46).

[0070] FIG. 44, presents the sequence of RT.opt.SF2 (native) (SEQ ID NO:47).

[0071] FIG. 45, presents the sequence of RTmut.SF2 (SEQ ID NO:48).

[0072] FIG. 46, presents the sequence of tat.exon1.sub.--2.opt.C22-37.SF2 (SEQ ID NO:49).

[0073] FIG. 47, presents the sequence of tat.exon1.sub.--2.opt.C37.SF2 (SEQ ID NO:50).

[0074] FIG. 48, presents the sequence of TatRevNef.opt.native.SF162 (SEQ ID NO:51).

[0075] FIG. 49, presents the sequence of TatRevNef.opt.SF162 (SEQ ID NO:52).

[0076] FIG. 50, presents the sequence of TatRevNefGag B (SEQ ID NO:53).

[0077] FIG. 51, sheets 1 and 2, presents the sequence of TatRevNefgagCpolIna B (SEQ ID NO:54).

[0078] FIG. 52, sheets 1 and 2, presents the sequence of TatRevNefGagProtInaRTmut B (SEQ ID NO:55).

[0079] FIG. 53, presents the sequence of TatRevNefp2Pol.opt_B (SEQ ID NO:56).

[0080] FIG. 54, presents the sequence of TatRevNefprotRTopt B (SEQ ID NO:57).

[0081] FIG. 55, presents the sequence of vif.opt.SF2 (SEQ ID NO:58).

[0082] FIG. 56, presents the sequence of vpr.opt.SF2 (SEQ ID NO:59).

[0083] FIG. 57, presents the sequence of vpu.opt.SF162 (SEQ ID NO:60).

[0084] FIG. 58, presents the sequence of gp140modSF162.GM135-154-186-195 (SEQ ID NO:61).

[0085] FIG. 59, presents the sequence of gp140modSF162.GM154 (SEQ ID NO:62).

[0086] FIG. 60, presents the sequence of gp140modSF162.GM154-186-195 (SEQ ID NO:63).

[0087] FIG. 61, presents the sequence of gp140mut7.modSF162.GM154 (SEQ ID NO:64).

[0088] FIG. 62 depicts alignment of amino acid sequences of various Env glycosylation mutants (GM), including amino acid translation of gp140modSF162 (SEQ ID NO:65); translation of gp140.modSF162.GM154 (SEQ ID NO:66); translation of gp140.modSF162.GM154-186-195 (SEQ ID NO:67); and gp140.modSF162.GM135-154-186-195 (SEQ ID NO:68).

[0089] FIG. 63 presents an overview of genome organization of HIV-1 and useful subgenomic fragments.

[0090] FIG. 64 presents antibody titer data from immunized rabbits following immunization with HIV Envelope DNA constructs and protein.

[0091] FIG. 65 presents a comparison of ELISA titers against subtype B and C Envelope proteins in rabbit sera collected after three DNA immunizations and a single protein boost.

[0092] FIG. 66 presents data of neutralizing antibody responses against subtype B SF162 EnvdV2 strain in rabbits immunized with subtype C TV1 Env in a DNA prime protein boost regimen.

[0093] FIG. 67 presents data of neutralizing antibody responses against subtype C primary strains, TV1 and TV2 in 5.25 reporter cell assay after a single protein boost.

[0094] FIG. 68 presents data of neutralizing antibody responses against subtype C, TV1 and Du174, and subtype B, SF162 after a single protein boost (as measured by Duke PBMC assay).

DETAILED DESCRIPTION OF THE INVENTION

[0095] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag).

[0096] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0097] As used in this specification, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a mixture of two or more such agents.

[0098] 1. Definitions

[0099] In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

[0100] "Synthetic" sequences, as used herein, refers to HIV polypeptide-encoding polynucleotides whose expression has been modified as described herein, for example, by codon substitution, altered activities, and/or inactivation of inhibitory sequences. "Wild-type" or "native" sequences, as used herein, refers to polypeptide encoding sequences that are essentially as they are found in nature, e.g., Gag, Pol, Vif, Vpr, Tat, Rev, Vpu, Env and/or Nef encoding sequences as found in HIV isolates, e.g., SF162, SF2, AF110965, AF110967, AF110968, AF110975, 8.sub.--5_TV1_C.ZA, 8.sub.--2_TV1_C.ZA or 12-5.sub.--1_TV2_C.ZA. The various regions of the HIV genome are shown in Table A, with numbering relative to 8.sub.--5_TV1C.ZA (FIGS. 1A-1D). Thus, the term "Pol" refers to one or more of the following polypeptides: polymerase (p6Pol); protease (prot); reverse transcriptase (p66RT or RT); RNAseH (p15RNAseH); and/or integrase (p31Int or Int). Identification of gene regions for any selected HIV isolate can be performed by one of ordinary skill in the art based on the teachings presented herein and the information known in the art, for example, by performing alignments relative to 8.sub.--5_TV1_C.ZA (FIGS. 1A-1D) or alignment to other known HIV isolates, for example, Subtype B lates with gene regions (e.g., SF2, GenBank Accession number K02007; SF162, GenBank Accession Number M38428, both herein incorporated by reference) and Subtype C isolates with gene regions (e.g., GenBank Accession Number AF110965 and GenBank Accession Number AF110975, both herein incorporated by reference).

[0101] As used herein, the term "virus-like particle" or "VLP" refers to a nonreplicating, viral shell, derived from any of several viruses discussed further below. VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art and discussed more fully below. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, X-ray crystallography, and the like. See, e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994) 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.

[0102] By "particle-forming polypeptide" derived from a particular viral protein is meant a full-length or near full-length viral protein, as well as a fragment thereof, or a viral protein with internal deletions, which has the ability to form VLPs under conditions that favor VLP formation. Accordingly, the polypeptide may comprise the full-length sequence, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. The term therefore intends deletions, additions and substitutions to the sequence, so long as the polypeptide retains the ability to form a VLP. Thus, the term includes natural variations of the specified polypeptide since variations in coat proteins often occur between viral isolates. The term also includes deletions, additions and substitutions that do not naturally occur in the reference protein, so long as the protein retains the ability to form a VLP. Preferred substitutions are those which are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic--aspartate and glutamate; (2) basic--lysine, arginine, histidine; (3) non-polar--alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar--glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.

[0103] The term "HIV polypeptide" refers to any amino acid sequence that exhibits sequence homology to native HIV polypeptides (e.g., Gag, Env, Prot, Pol, RT, Int, vif, vpr, vpu, tat, rev, nef and/or combinations thereof) and/or which is functional. Non-limiting examples of functions that may be exhibited by HIV polypeptides include, use as immunogens (e.g., to generate a humoral and/or cellular immune response), use in diagnostics (e.g, bound by suitable antibodies for use in ELISAs or other immunoassays) and/or polypeptides which exhibit one or more biological activities associated with the wild type or synthetic HIV polypeptide. For example, as used herein, the term "Gag polypeptide" may refer to a polypeptide that is bound by one or more anti-Gag antibodies; elicits a humoral and/or cellular immune response; and/or exhibits the ability to form particles.

[0104] An "antigen" refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term "immunogen." Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term "antigen" denotes both subunit antigens, (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as, killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes. Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. Similarly, an oligonucleotide or polynucleotide which expresses an antigen or antigenic determinant in vivo, such as in gene therapy and DNA immunization applications, is also included in the definition of antigen herein.

[0105] For purposes of the present invention, antigens can be derived from any of several known viruses, bacteria, parasites and fungi, as described more fully below. The term also intends any of the various tumor antigens. Furthermore, for purposes of the present invention, an "antigen" refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.

[0106] An "immunological response" to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells ("CTL"s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.

[0107] A composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an immunized host.

[0108] The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (e.g., by the tetramer technique)(reviewed by McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med. 187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev. 150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

[0109] Thus, an immunological response as used herein may be one which stimulates the production of CTLs, and/or the production or activation of helper T-cells. The antigen of interest may also elicit an antibody-mediated immune response. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or .gamma..delta. T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.

[0110] An "immunogenic composition" is a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest. The immunogenic composition can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal and mucosal (e.g., intra-rectally or intra-vaginally) administration.

[0111] By "subunit vaccine" is meant a vaccine composition which includes one or more selected antigens but not all antigens, derived from or homologous to, an antigen from a pathogen of interest such as from a virus, bacterium, parasite or fungus. Such a composition is substantially free of intact pathogen cells or pathogenic particles, or the lysate of such cells or particles. Thus, a "subunit vaccine" can be prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or analogs thereof. The method of obtaining an antigen included in the subunit vaccine can thus include standard purification techniques, recombinant production, or synthetic production.

[0112] "Substantially purified" general refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0113] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence such as a stop codon may be located 3' to the coding sequence.

[0114] Typical "control elements", include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences. For example, the sequences and/or vectors described herein may also include one or more additional sequences that may optimize translation and/or termination including, but not limited to, a Kozak sequence (e.g., GCCACC placed in front (5') of the ATG of the codon-optimized wild-type leader or any other suitable leader sequence (e.g., tpa1, tpa2, wtLnat (native wild-type leader)) or a termination sequence (e.g., TAA or, preferably, TAAA placed after (3') the coding sequence.

[0115] A "polynucleotide coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are determined by a start codon, for example, at or near the 5' terminus and a translation stop codon, for example, at or near the 3' terminus. Exemplary coding sequences are the modified viral polypeptide-coding sequences of the present invention. The coding regions of the polynucleotide sequences of the present invention are identifiable by one of skill in the art and may, for example, be easily identified by performing translations of all three frames of the polynucleotide and identifying the frame corresponding to the encoded polypeptide, for example, a synthetic nef polynucleotide of the present invention encodes a nef-derived polypeptide. A transcription termination sequence may be located 3' to the coding sequence. Typical "control elements", include, but are not limited to, transcription regulators, such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences; and translation regulators, such as sequences for optimization of initiation of translation, e.g., Shine-Dalgarno (ribosome binding site) sequences, Kozak sequences (i.e., sequences for the optimization of translation, located, for example, 5' to the coding sequence), leader sequences, translation initiation codon (e.g., ATG), and translation termination sequences. In certain embodiments, one or more translation regulation or initiation sequences (e.g., the leader sequence) are derived from wild-type translation initiation sequences, i.e., sequences that regulate translation of the coding region in their native state. Wild-type leader sequences that have been modified, using the methods described herein, also find use in the present invention. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.

[0116] A "nucleic acid" molecule can include, but is not limited to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.

[0117] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0118] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. "Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used inter-changeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.

[0119] Techniques for determining amino acid sequence "similarity" are well known in the art. In general, "similarity" means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" then can be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.

[0120] Two or more polynucleotide sequences can be compared by determining their "percent identity." Two or more amino acid sequences likewise can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or peptide sequences, is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An implementation of this algorithm for nucleic acid and peptide sequences is provided by the Genetics Computer Group (Madison, Wis.) in their BestFit utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). Other equally suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

[0121] For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions. Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated, the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, such as the alignment program BLAST, which can also be used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0122] One of skill in the art can readily determine the proper search parameters to use for a given sequence, exemplary preferred Smith Waterman based parameters are presented above. For example, the search parameters may vary based on the size of the sequence in question. Thus, for the polynucleotide sequences of the present invention the length of the polynucleotide sequence disclosed herein is searched against a selected database and compared to sequences of essentially the same length to determine percent identity. For example, a representative embodiment of the present invention would include an isolated polynucleotide comprising X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least about a selected level of percent identity relative to Y contiguous nucleotides of one or more of the sequences described herein (e.g., in Table C) or fragment thereof, and (ii) for search purposes X equals Y, wherein Y is a selected reference polynucleotide of defined length (for example, a length of from 15 nucleotides up to the number of nucleotides present in a selected full-length sequence).

[0123] The sequences of the present invention can include fragments of the sequences, for example, from about 15 nucleotides up to the number of nucleotides present in the full-length sequences described herein (e.g., see the Figures), including all integer values falling within the above-described range. For example, fragments of the polynucleotide sequences of the present invention may be 30-60 nucleotides, 60-120 nucleotides, 120-240 nucleotides, 240-480 nucleotides, 480-1000 nucleotides, and all integer values therebetween.

[0124] The synthetic expression cassettes (and purified polynucleotides) of the present invention include related polynucleotide sequences having about 80% to 100%, greater than 80-85%, preferably greater than 90-92%, more preferably greater than 95%, and most preferably greater than 98% up to 100% (including all integer values falling within these described ranges) sequence identity to the synthetic expression cassette and/or polynucleotide sequences disclosed herein (for example, to the sequences of the present invention) when the sequences of the present invention are used as the query sequence against, for example, a database of sequences.

[0125] Two nucleic acid fragments are considered to "selectively hybridize" as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., supra or Ausubel et al., supra). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.

[0126] When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent" typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

[0127] With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., supra or Ausubel et al., supra).

[0128] A first polynucleotide is "derived from" second polynucleotide if it has the same or substantially the same basepair sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above.

[0129] A first polypeptide is "derived from" a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.

[0130] Generally, a viral polypeptide is "derived from" a particular polypeptide of a virus (viral polypeptide) if it is (i) encoded by an open reading frame of a polynucleotide of that virus (viral polynucleotide), or (ii) displays sequence identity to polypeptides of that virus as described above.

[0131] "Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence. Further, polyproteins can be constructed by fusing in-frame two or more polynucleotide sequences encoding polypeptide or peptide products. Further, polycistronic coding sequences may be produced by placing two or more polynucleotide sequences encoding polypeptide products adjacent each other, typically under the control of one promoter, wherein each polypeptide coding sequence may be modified to include sequences for internal ribosome binding sites.

[0132] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0133] By "nucleic acid immunization" is meant the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell, for the in vivo expression of an antigen, antigens, an epitope, or epitopes. The nucleic acid molecule can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal and mucosal administration, or the like, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the antigen encoded by the nucleic acid molecule.

[0134] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from alphaviruses, pox viruses and vaccinia viruses. When used for immunization, such gene delivery expression vectors may be referred to as vaccines or vaccine vectors.

[0135] "T lymphocytes" or "T cells" are non-antibody producing lymphocytes that constitute a part of the cell-mediated arm of the immune system. T cells arise from immature lymphocytes that migrate from the bone marrow to the thymus, where they undergo a maturation process under the direction of thymic hormones. Here, the mature lymphocytes rapidly divide increasing to very large numbers. The maturing T cells become immunocompetent based on their ability to recognize and bind a specific antigen. Activation of immunocompetent T cells is triggered when an antigen binds to the lymphocyte's surface receptors.

[0136] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0137] A "vector" is capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0138] Transfer of a "suicide gene" (e.g., a drug-susceptibility gene) to a target cell renders the cell sensitive to compounds or compositions that are relatively nontoxic to normal cells. Moolten, F. L. (1994) Cancer Gene Ther. 1:279-287. Examples of suicide genes are thymidine kinase of herpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996) Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al. (1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosine deaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells which express these genes are rendered sensitive to the effects of the relatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide (cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase) or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992) Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA 91:8302-8306.

[0139] A "selectable marker" or "reporter marker" refers to a nucleotide sequence included in a gene transfer vector that has no therapeutic activity, but rather is included to allow for simpler preparation, manufacturing, characterization or testing of the gene transfer vector.

[0140] A "specific binding agent" refers to a member of a specific binding pair of molecules wherein one of the molecules specifically binds to the second molecule through chemical and/or physical means. One example of a specific binding agent is an antibody directed against a selected antigen.

[0141] By "subject" is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as rhesus macaque, chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The system described above is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

[0142] By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0143] By "physiological pH" or a "pH in the physiological range" is meant a pH in the range of approximately 7.0 to 8.0 inclusive, more typically in the range of approximately 7.2 to 7.6 inclusive.

[0144] As used herein, "treatment" refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).

[0145] By "co-administation" is meant administation of more than one compostion (e.g., multiple or molecule. Thus, co-administration includes concurrent administration or sequentially administration (in any order), via the same or different routes of administration. Non-limiting example of co-administration regimes include, co-administration of nucleic acid and polypeptide; co-administration of different nucleic acids (e.g., different expression cassettes as described herein and/or different gene delivery vectors); and co-dministration of different polypeptides (e.g., different HIV polypeptides and/or different adjuvants). The term also encompasses multiple administration of one of the co-administered molecules or compositions (e.g., multiple administration of one or more of the expression cassettes described herein followed by one or more administration of a polypeptide-containing composition). In cases where the molecules or composions are delivered sequentially, the time between each administration can be readily determined by one of skill in the art in view of the teaching herein. "Lentiviral vector", and "recombinant lentiviral vector" refer to a nucleic acid construct which carries, and within certain embodiments, is capable of directing the expression of a nucleic acid molecule of interest. The lentiviral vector include at least one transcriptional promoter/enchancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transciptional modification of protein. Such vector constructs must also include a packaging signal, long terminal repeats (LTRS) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector). Optionally, the recombinant lentiviral vector may also include a signal which directs polyadenylation, selectable markers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence. By way of example, such vectors typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3.zeta.LTR or a portion thereof

[0146] "Lentiviral vector particle" as utilized within the present invention refers to a lentivirus which carries at least one gene of interest. The retrovirus may also contain a selectable marker. The recombinant lentivirus is capable of reverse transcribing its genetic material (RNA) into DNA and incorporating this genetic material into a host cell's DNA upon infection. Lentiviral vector particles may have a lentiviral envelope, a non-lentiviral envelope (e.g., an ampho or VSV-G envelope), or a chimeric envelope.

[0147] "Nucleic acid expression vector" or "Expression cassette" refers to an assembly which is capable of directing the expression of a sequence or gene of interest. The nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. Expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0148] "Packaging cell" refers to a cell which contains those elements necessary for production of infectious recombinant retrovirus which are lacking in a recombinant retroviral vector. Typically, such packaging cells contain one or more expression cassettes which are capable of expressing proteins which encode Gag, pol and env proteins.

[0149] "Producer cell" or "vector producing cell" refers to a cell which contains all elements necessary for production of recombinant retroviral vector particles.

[0150] 2. Modes of Carrying Out the Invention

[0151] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0152] Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

[0153] 2.1. The HIV Genome

[0154] The HIV genome and various polypeptide-encoding regions are shown in Table A. The nucleotide positions are given relative to 8.sub.--5_TV1_C.ZA (FIG. 1; an HIV Type C isolate). However, it will be readily apparent to one of ordinary skill in the art in view of the teachings of the present disclosure how to determine corresponding regions in other HIV strains or variants (e.g., isolates HIV.sub.IIIb, HIV.sub.SF2, HIV-1.sub.SF162, HIV-1.sub.SF170, HIV.sub.LAV, HIV.sub.LAI, HIV.sub.MN, HIV-1.sub.CM235, HIV-1.sub.US4, other HIV-1 strains from diverse subtypes (e.g., subtypes, A through G, and O), HIV-2 strains and diverse subtypes (e.g., HIV-2.sub.UC1 and HIV-2.sub.UC2), and simian immunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.; for a description of these and other related viruses), using for example, sequence comparison programs (e.g., BLAST and others described herein) or identification and alignment of structural features (e.g., a program such as the "ALB" program described herein that can identify the various regions). TABLE-US-00001 TABLE A Regions of the HIV Genome relative to 8_5_TV1_C.ZA Region Position in nucleotide sequence 5'LTR 1-636 U3 1-457 R 458-553 U5 554-636 NFkB II 340-348 NFkB I 354-362 Sp1 III 379-388 Sp1 II 390-398 Sp1 I 400-410 TATA Box 429-433 TAR 474-499 Poly A signal 529-534 PBS 638-655 p7 binding region, packaging signal 685-791 Gag: 792-2285 p17 792-1178 p24 1179-1871 Cyclophilin A bdg. 1395-1505 MHR 1632-1694 p2 1872-1907 p7 1908-2072 Frameshift slip 2072-2078 p1 2073-2120 p6Gag 2121-2285 Zn-motif I 1950-1991 Zn-motif II 2013-2054 Pol: 2072-5086 p6Pol 2072-2245 Prot 2246-2542 p66RT 2543-4210 p15RNaseH 3857-4210 p31Int 4211-5086 Vif: 5034-5612 Hydrophilic region 5292-5315 Vpr: 5552-5839 Oligomerization 5552-5677 Amphipathic a-helix 5597-5653 Tat: 5823-6038 and 8417-8509 Tat-1 exon 5823-6038 Tat-2 exon 8417-8509 N-terminal domain 5823-5885 Trans-activation domain 5886-5933 Transduction domain 5961-5993 Rev: 5962-6037 and 8416-8663 Rev-1 exon 5962-6037 Rev-2 exon 8416-8663 High-affinity bdg. site 8439-8486 Leu-rich effector domain 8562-8588 Vpu: 6060-6326 Transmembrane domain 6060-6161 Cytoplasmic domain 6162-6326 Env (gp160): 6244-8853 Signal peptide 6244-6324 gp120 6325-7794 V1 6628-6729 V2 6727-6852 V3 7150-7254 V4 7411-7506 V5 7663-7674 C1 6325-6627 C2 6853-7149 C3 7255-7410 C4 7507-7662 C5 7675-7794 CD4 binding 7540-7566 gp41 7795-8853 Fusion peptide 7789-7842 Oligomerization domain 7924-7959 N-terminal heptad repeat 7921-8028 C-terminal heptad repeat 8173-8280 Immunodominant region 8023-8076 Nef: 8855-9478 Myristoylation 8858-8875 SH3 binding 9062-9091 Polypurine tract 9128-9154 SH3 binding 9296-9307

[0155] It will be readily apparent that one of skill in the art can readily align any sequence to that shown in Table A to determine relative locations of any particular HIV gene. For example, using one of the alignment programs described herein (e.g., BLAST), other HIV genomic sequences can be aligned with 8.sub.--5_TV1_C.ZA (Table A) and locations of genes determined. Polypeptide sequences can be similarly aligned. For example, FIGS. 2A-2C shows the alignment of Env polypeptide sequences from various strains, relative to SF-162. As described in detail in co-owned WO/39303 (herein incorporated by reference), Env polypeptides (e.g., gp120, gp140 and gp160) include a "bridging sheet" comprised of 4 anti-parallel b-strands (b-2, b-3, b-20 and b-21) that form a b-sheet. Extruding from one pair of the b-strands (b-2 and b-3) are two loops, V1 and V2. The b-2 sheet occurs at approximately amino acid residue 113 (Cys) to amino acid residue 117 (Thr) while b-3 occurs at approximately amino acid residue 192 (Ser) to amino acid residue 194 (Ile), relative to SF-162. The "V1/V2 region" occurs at approximately amino acid positions 120 (Cys) to residue 189 (Cys), relative to SF-162. Extruding from the second pair of b-strands (b-20 and b-21) is a "small-loop" structure, also referred to herein as "the bridging sheet small loop." The locations of both the small loop and bridging sheet small loop can be determined relative to HXB-2 following the teachings herein and in WO/39303. Also shown by arrows in FIG. 2A-C are approximate sites for deletions sequence from the beta sheet region. The "*" denotes N-glycosylation sites that can be mutated following the teachings of the present specification.

[0156] 2.2.0 Synthetic Expression Cassettes

[0157] One aspect of the present invention is the generation of HIV-1 coding sequences, and related sequences, for example having improved expression relative to the corresponding wild-type sequences.

[0158] 2.2.1 Modification of HIV-1 Nucleic Acid Coding Sequences

[0159] First, the HIV-1 codon usage pattern was modified so that the resulting nucleic acid coding sequence was comparable to codon usage found in highly expressed human genes. The HIV codon usage reflects a high content of the nucleotides A or T of the codon-triplet. The effect of the HIV-1 codon usage is a high AT content in the DNA sequence that results in a decreased translation ability and instability of the mRNA. In comparison, highly expressed human codons prefer the nucleotides G or C. The HIV coding sequences were modified to be comparable to codon usage found in highly expressed human genes.

[0160] Second, there are inhibitory (or instability) elements (INS) located within the coding sequences of, for example, the Gag coding sequences. The RRE is a secondary RNA structure that interacts with the HIV encoded Rev-protein to overcome the expression down-regulating effects of the INS. To overcome the post-transcriptional activating mechanisms of RRE and Rev, the instability elements can be inactivated by introducing multiple point mutations that do not alter the reading frame of the encoded proteins.

[0161] Third, for some genes the coding sequence has been altered such that the polynucleotide coding sequence encodes a gene product that is inactive or non-functional (e.g., inactivated polymerase, protease, tat, rev, nef, vif, vpr, and/or vpu gene products). Example 1 describes some exemplary mutations. Example 8 presents information concerning functional analysis of mutated Tat, Rev and Nef antigens.

[0162] The synthetic coding sequences are assembled by methods known in the art, for example by companies such as the Midland Certified Reagent Company (Midland, Tex.).

[0163] Modification of the Gag polypeptide coding sequences results in improved expression relative to the wild-type coding sequences in a number of mammalian cell lines (as well as other types of cell lines, including, but not limited to, insect cells).

[0164] Some exemplary polynucleotide sequences encoding Gag-containing polypeptides are GagComplPolmut.SF2, GagComplPolmutAtt.SF2, GagComplPolmutIna.SF2, gagCpolInaTatRevNef.opt_B, GagPolmutAtt.SF2, GagPolmutIna.SF2, GagProtInaRTmut.SF2, GagProtInaRTmutTatRevNef.opt_B, GagRTmut.SF2, and GagTatRevNef.opt_B.

[0165] Similarly, the present invention also includes synthetic Env-encoding polynucleotides and modified Env proteins, for example, gp140.modSF162.CwtLmod, gp140.modSF162.CwtLnat, gp160.modSF162.delV2.mut7, and gp160.modSF162.delV2.mut8.

[0166] The codon usage pattern for Env was modified as described above for Gag so that the resulting nucleic acid coding sequence was comparable to codon usage found in highly expressed human genes. Experiments performed in support of the present invention show that the synthetic Env sequences were capable of higher level of protein production relative to the native Env sequences.

[0167] Modification of the Env polypeptide coding sequences results in improved expression relative to the wild-type coding sequences in a number of mammalian cell lines (as well as other types of cell lines, including, but not limited to, insect cells). Similar Env polypeptide coding sequences can be obtained, modified and tested for improved expression from a variety of isolates, including those described above for Gag.

[0168] Further modifications of Env include, but are not limited to, generating polynucleotides that encode Env polypeptides having mutations and/or deletions therein. For instance, the hypervariable regions, V1 and/or V2, can be deleted as described herein. Additionally, other modifications, for example to the bridging sheet region and/or to N-glycosylation sites within Env can also be performed following the teachings of the present specification. (see, FIG. 2A-C, as well as WO 00/39303, WO 00/39302, WO 00/39304, WO 02/04493 all herein incorporated by reference in their entireties). Various combinations of these modifications can be employed to generate synthetic expression cassettes as described herein.

[0169] The present invention also includes expression cassettes which include synthetic Pol sequences. As noted above, "Pol" includes, but is not limited to, the protein-encoding regions comprising polymerase, protease, reverse transcriptase and/or integrase-containing sequences (Wan et et al (1996) Biochem. J. 316:569-573; Kohl et al. (1988) PNAS USA 85:4686-4690; Krausslich et al. (1988) J. Virol. 62:4393-4397; Coffin, "Retroviridae and their Replication" in Virology, pp 1437-1500 (Raven, New York, 1990); Patel et. al. (1995) Biochemistry 34:5351-5363). Thus, the synthetic expression cassettes exemplified herein include one or more of these regions and one or more changes to the resulting amino acid sequences. Some exemplary polynucleotide sequences encoding Pol-derived polypeptides are presented in Table C.

[0170] The codon usage pattern for Pol was modified as described above for Gag and Env so that the resulting nucleic acid coding sequence was comparable to codon usage found in highly expressed human genes.

[0171] Constructs may be modified in various ways. For example, the expression constructs may include a sequence that encodes the first 6 amino acids of the integrase polypeptide. This 6 amino acid region is believed to provide a cleavage recognition site recognized by HIV protease (see, e.g., McCornack et al. (1997) FEBS Letts 414:84-88). Constructs may include a multiple cloning site (MCS) for insertion of one or more transgenes, typically at the 3' end of the construct. In addition, a cassette encoding a catalytic center epitope derived from the catalytic center in RT is typically included 3' of the sequence encoding 6 amino acids of integrase. This cassette encodes Ile178 through Serine 191 of RT and may be added to keep this well conserved region as a possible CTL epitope. Further, the constructs contain an insertion mutations to preserve the reading frame. (see, e.g., Park et al. (1991) J. Virol. 65:5111).

[0172] In certain embodiments, the catalytic center and/or primer grip region of RT are modified. The catalytic center and primer grip regions of RT are described, for example, in Patel et al. (1995) Biochem. 34:5351 and Palaniappan et al. (1997) J. Biol. Chem. 272(17): 11157. For example, wild type sequence encoding the amino acids YMDD at positions 183-185 of p66 RT, numbered relative to AF110975, may be replaced with sequence encoding the amino acids "AP". Further, the primer grip region (amino acids WMGY, residues 229-232 of p66RT, numbered relative to AF110975) may be replaced with sequence encoding the amino acids "PI."

[0173] For the Pol sequence, the changes in codon usage are typically restricted to the regions up to the -1 frameshift and starting again at the end of the Gag reading frame; however, regions within the frameshift translation region can be modified as well. Finally, inhibitory (or instability) elements (INS) located within the coding sequences of the protease polypeptide coding sequence can be altered as well.

[0174] Experiments can be performed in support of the present invention to show that the synthetic Pol sequences were capable of higher level of protein production relative to the native Pol sequences. Modification of the Pol polypeptide coding sequences results in improved expression relative to the wild-type coding sequences in a number of mammalian cell lines (as well as other types of cell lines, including, but not limited to, insect cells). Similar Pol polypeptide coding sequences can be obtained, modified and tested for improved expression from a variety of isolates, including those described above for Gag and Env.

[0175] The present invention also includes expression cassettes which include synthetic sequences derived HIV genes other than Gag, Env and Pol, including but not limited to, regions within Gag, Env, Pol, as well as, GagComplPolmut.SF2, GagComplPolmutAtt.SF2, GagComplPolmutIna.SF2, gagCpolInaTatRevNef.opt_B, GagPolmutAtt.SF2, GagPolmutIna.SF2, GagProtInaRTmut.SF2, GagProtInaRTmutTatRevNef.opt_B, GagRTmut.SF2, GagTatRevNef.opt_B, gp140.modSF162.CwtLmod, gp140.modSF162.CwtLnat, gp160.modSF162.delV2.mut7, gp160.modSF162.delV2.mut8, int.opt.mut.SF2, int.opt.SF2, nef.D125G.-myr.opt.SF162, nef.D107G.-myr18.opt.SF162, nef.opt.D125G.SF162, nef.opt.SF162, p15RnaseH.opt.SF2, p2Pol.opt.YMWM.SF2, p2PolInaopt.YM.SF2, p2Polopt.SF2, p2PolTatRevNef.opt.native_B, p2PolTatRevNef.opt_B, pol.opt.SF2, prot.opt.SF2, protIna.opt.SF2, protInaRT.YM.opt.SF2, protInaRT.YMWM.opt.SF2, ProtInaRTmut.SF2, protRT.opt.SF2, ProtRT.TatRevNef.opt_B, ProtRTTatRevNef.opt_B, rev.exon1.sub.--2.M5-10.opt.SF162, rev.exon1.sub.--2.opt.SF162, RT.opt.SF2 (mutant), RT.opt.SF2 (native), RTmut.SF2, tat.exon1.sub.--2.opt.C22-37.SF2, tat.exon1.sub.--2.opt.C37.SF2, TatRevNef.opt.native.SF162, TatRevNef.opt.SF162, TatRevNefGag B, TatRevNefgagCpolIna B, TatRevNefGagProtInaRTmut B, TatRevNefp2Pol.opt_B, TatRevNefprotRTopt B, vif.opt.SF2, vpr.opt.SF2, and vpu.opt.SF162. Sequences obtained from other strains can be manipulated in similar fashion following the teachings of the present specification. As noted above, the codon usage pattern is modified as described above for Gag, Env and Pol so that the resulting nucleic acid coding sequence is comparable to codon usage found in highly expressed human genes. Typically these synthetic sequences are capable of higher level of protein production relative to the native sequences and that modification of the wild-type polypeptide coding sequences results in improved expression relative to the wild-type coding sequences in a number of mammalian cell lines (as well as other types of cell lines, including, but not limited to, insect cells). Furthermore, the nucleic acid sequence can also be modified to introduce mutations into one or more regions of the gene, for instance to alter the function of the gene product (e.g., render the gene product non-functional) and/or to eliminate site modifications (e.g., the myristoylation site in Nef).

[0176] Synthetic expression cassettes, derived from HIV Type B coding sequences, exemplified herein include, but are not limited to, those comprising one or more of the following synthetic polynucleotides: GagComplPolmut.SF2, GagComplPolmutAtt.SF2, GagComplPolmutIna.SF2, gagCpolInaTatRevNef.opt_B, GagPolmutAtt.SF2, GagPolmutIna.SF2, GagProtInaRTmut.SF2, GagProtInaRTmutTatRevNef.opt_B, GagRTmut.SF2, GagTatRevNef.opt_B, gp140.modSF162.CwtLmod, gp140.modSF162.CwtLnat, gp160.modSF162.delV2.mut7, gp160.modSF162.delV2.mut8, int.opt.mut.SF2, int.opt.SF2, nef.D125G.-myr.opt.SF162, nef.D107G.-myr18.opt.SF162, nef.opt.D125G.SF162, nef.opt.SF162, p15RnaseH.opt.SF2, p2Pol.opt.YMWM.SF2, p2PolInaopt.YM.SF2, p2Polopt.SF2, p2PolTatRevNef.opt.native_B, p2PolTatRevNef.opt_B, pol.opt.SF2, prot.opt.SF2, protIna.opt.SF2, protInaRT.YM.opt.SF2, protInaRT.YMWM.opt.SF2, ProtInaRTmut.SF2, protRT.opt.SF2, ProtRT.TatRevNef.opt_B, ProtRTTatRevNef.opt_B, rev.exon1.sub.--2.M5-10.opt.SF162, rev.exon1.sub.--2.opt.SF162, RT.opt.SF2 (mutant), RT.opt.SF2 (native), RTmut.SF2, tat.exon1.sub.--2.opt.C22-37.SF2, tat.exon1.sub.--2.opt.C37.SF2, TatRevNef.opt.native.SF162, TatRevNef.opt.SF162, TatRevNefGag B, TatRevNefgagCpolIna B, TatRevNefGagProtInaRTmut B, TatRevNefp2Pol.opt_B, TatRevNefprotRTopt B, vif.opt.SF2, vpr.opt.SF2, and vpu.opt.SF162.

[0177] Gag-complete refers to in-frame polyproteins comprising, e.g., Gag and pol, wherein the p6 portion of Gag is present.

[0178] Additional sequences that may be employed in some aspects of the present invention have been described in WO 00/39302, WO 00/39303, WO 00/39304, and WO 02/04493, all of which are herein incorporated by reference in their entireties.

[0179] 2.2.2 Further Modification of Sequences Including HIV Nucleic Acid Coding Sequences

[0180] The HIV polypeptide-encoding expression cassettes described herein may also contain one or more further sequences encoding, for example, one or more transgenes. Further sequences (e.g., transgenes) useful in the practice of the present invention include, but are not limited to, further sequences are those encoding further viral epitopes/antigens {including but not limited to, HCV antigens (e.g., E1, E2; Houghton, M., et al., U.S. Pat. No. 5,714,596, issued Feb. 3, 1998; Houghton, M., et al., U.S. Pat. No. 5,712,088, issued Jan. 27, 1998; Houghton, M., et al., U.S. Pat. No. 5,683,864, issued Nov. 4, 1997; Weiner, A. J., et al., U.S. Pat. No. 5,728,520, issued Mar. 17, 1998; Weiner, A. J., et al., U.S. Pat. No. 5,766,845, issued Jun. 16, 1998; Weiner, A. J., et al., U.S. Pat. No. 5,670,152, issued Sep. 23, 1997; all herein incorporated by reference), HIV antigens (e.g., derived from one or more HIV isolate); and sequences encoding tumor antigens/epitopes. Further sequences may also be derived from non-viral sources, for instance, sequences encoding cytokines such interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1I), interleukin-11 (IL-11), MIP-1I, tumor necrosis factor (TNF), leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand, commercially available from several vendors such as, for example, Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems and Immunex (Seattle, Wash.). Additional sequences are described below. Also, variations on the orientation of the Gag and other coding sequences, relative to each other, are described below.

[0181] HIV polypeptide coding sequences can be obtained from other HIV isolates, see, e.g., Myers et al. Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al., Human Retroviruses and Aids, 1997, Los Alamos, N. Mex.: Los Alamos National Laboratory. Synthetic expression cassettes can be generated using such coding sequences as starting material by following the teachings of the present specification.

[0182] Further, the synthetic expression cassettes of the present invention include related polypeptide sequences having greater than 85%, preferably greater than 90%, more preferably greater than 95%, and most preferably greater than 98% sequence identity to the polypeptides encoded by the synthetic expression cassette sequences disclosed herein.

[0183] Exemplary expression cassettes and modifications are set forth in Example 1.

[0184] 2.2.3 Expression of Synthetic Sequences Encoding HIV-1 Polypeptides and Related Polypeptides

[0185] Synthetic HIV-encoding sequences (expression cassettes) of the present invention can be cloned into a number of different expression vectors to evaluate levels of expression and, in the case of Gag-containing constructs, production of VLPs. The synthetic DNA fragments for HIV polypeptides can be cloned into eucaryotic expression vectors, including, a transient expression vector, CMV-promoter-based mammalian vectors, and a shuttle vector for use in baculovirus expression systems. Corresponding wild-type sequences can also be cloned into the same vectors.

[0186] These vectors can then be transfected into a several different cell types, including a variety of mammalian cell lines (293, RD, COS-7, and CHO, cell lines available, for example, from the A.T.C.C.). The cell lines are then cultured under appropriate conditions and the levels of any appropriate polypeptide product can be evaluated in supernatants. (see, Table A). For example, p24 can be used to evaluate Gag expression; gp160, gp140 or gp120 can be used to evaluate Env expression; p6pol can be used to evaluate Pol expression; prot can be used to evaluate protease; p15 for RNAseH; p31 for Integrase; and other appropriate polypeptides for Vif, Vpr, Tat, Rev, Vpu and Nef. Further, modified polypeptides can also be used, for example, other Env polypeptides include, but are not limited to, for example, native gp160, oligomeric gp140, monomeric gp120 as well as modified and/or synthetic sequences of these polypeptides. The results of these assays demonstrate that expression of synthetic HIV polypeptide-encoding sequences are significantly higher than corresponding wild-type sequences.

[0187] Further, Western Blot analysis can be used to show that cells containing the synthetic expression cassette produce the expected protein at higher per-cell concentrations than cells containing the native expression cassette. The HIV proteins can be seen in both cell lysates and supernatants. The levels of production are significantly higher in cell supernatants for cells transfected with the synthetic expression cassettes of the present invention.

[0188] Fractionation of the supernatants from mammalian cells transfected with the synthetic expression cassette can be used to show that the cassettes provide superior production of HIV proteins and, in the case of Gag, VLPs, relative to the wild-type sequences.

[0189] Efficient expression of these HIV-containing polypeptides in mammalian cell lines provides the following benefits: the polypeptides are free of baculovirus contaminants; production by established methods approved by the FDA; increased purity; greater yields (relative to native coding sequences); and a novel method of producing the Sub HIV-containing polypeptides in CHO cells which is not feasible in the absence of the increased expression obtained using the constructs of the present invention. Exemplary Mammalian cell lines include, but are not limited to, BHK, VERO, HT1080, 293, 293T, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, and CEMX174 (such cell lines are available, for example, from the A.T.C.C.).

[0190] A synthetic Gag expression cassette of the present invention will also exhibit high levels of expression and VLP production when transfected into insect cells. Synthetic expression cassettes described herein also demonstrate high levels of expression in insect cells. Further, in addition to a higher total protein yield, the final product from the synthetic polypeptides consistently contains lower amounts of contaminating baculovirus proteins than the final product from the native sequences.

[0191] Further, synthetic expression cassettes of the present invention can also be introduced into yeast vectors which, in turn, can be transformed into and efficiently expressed by yeast cells (Saccharomyces cerevisea; using vectors as described in Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference).

[0192] In addition to the mammalian and insect vectors, the synthetic expression cassettes of the present invention can be incorporated into a variety of expression vectors using selected expression control elements. Appropriate vectors and control elements for any given cell an be selected by one having ordinary skill in the art in view of the teachings of the present specification and information known in the art about expression vectors.

[0193] For example, a synthetic expression cassette can be inserted into a vector which includes control elements operably linked to the desired coding sequence, which allow for the expression of the gene in a selected cell-type. For example, typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (a CMV promoter can include intron A), RSV, HIV-Ltr, the mouse mammary tumor virus LTR promoter (MMLV-ltr), the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook, et al., supra, as well as a bovine growth hormone terminator sequence. Introns, containing splice donor and acceptor sites, may also be designed into the constructs for use with the present invention (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986).

[0194] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986).

[0195] The desired synthetic polypeptide encoding sequences can be cloned into any number of commercially available vectors to generate expression of the polypeptide in an appropriate host system. These systems include, but are not limited to, the following: baculovirus expression {Reilly, P. R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames, et al., Biotechniques 11:378 (1991); Pharmingen; Clontech, Palo Alto, Calif.)}, vaccinia expression {Earl, P. L., et al., "Expression of proteins in mammalian cells using vaccinia" In Current Protocols in Molecular Biology (F. M. Ausubel, et al. Eds.), Greene Publishing Associates & Wiley Interscience, New York (1991); Moss, B., et al., U.S. Pat. No. 5,135,855, issued 4 Aug. 1992}, expression in bacteria {Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media Pa.; Clontech}, expression in yeast (Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated by reference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93 (1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D. V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink, Methods in Enzymology 194 (1991)}, expression in mammalian cells {Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary (CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983); 1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman, R. J., "Selection and coamplification of heterologous genes in mammalian cells," in Methods in Enzymology, vol. 185, pp 537-566. Academic Press, Inc., San Diego Calif. (1991)}, and expression in plant cells {plant cloning vectors, Clontech Laboratories, Inc., Palo Alto, Calif., and Pharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al., J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol. Lett. 67:325 (1990); An, et al., "Binary Vectors", and others in Plant Molecular Biology Manual A3: 1-19 (1988); Miki, B. L. A., et al., pp. 249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al., eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology: Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley, 1997; Miglani, Gurbachan Dictionary of Plant Genetics and Molecular Biology, New York, Food Products Press, 1998; Henry, R. J., Practical Applications of Plant Molecular Biology, New York, Chapman & Hall, 1997}.

[0196] Also included in the invention is an expression vector, containing coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements generally include a promoter, translation initiation codon, and translation and transcription termination sequences, and an insertion site for introducing the insert into the vector. Translational control elements have been reviewed by M. Kozak (e.g., Kozak, M., Mamm. Genome 7(8):563-574, 1996; Kozak, M., Biochimie 76(9):815-821, 1994; Kozak, M., J Cell Biol 108(2):229-241, 1989; Kozak, M., and Shatkin, A. J., Methods Enzymol 60:360-375, 1979).

[0197] Expression in yeast systems has the advantage of commercial production. Recombinant protein production by vaccinia and CHO cell line have the advantage of being mammalian expression systems. Further, vaccinia virus expression has several advantages including the following: (i) its wide host range; (ii) faithful post-transcriptional modification, processing, folding, transport, secretion, and assembly of recombinant proteins; (iii) high level expression of relatively soluble recombinant proteins; and (iv) a large capacity to accommodate foreign DNA.

[0198] The recombinantly expressed polypeptides from synthetic HIV polypeptide-encoding expression cassettes are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, gel filtration, size-exclusion chromatography, size-fractionation, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated based on, for example, HIV antigens.

[0199] Advantages of expressing the proteins of the present invention using mammalian cells include, but are not limited to, the following: well-established protocols for scale-up production; the ability to produce VLPs; cell lines are suitable to meet good manufacturing process (GMP) standards; culture conditions for mammalian cells are known in the art.

[0200] Synthetic HIV 1 polynucleotides are described herein, see, for example, the figures. Various forms of the different embodiments of the invention, described herein, may be combined.

[0201] Exemplary expression assays are set forth in Example 2. Exemplary conditions for Western Blot analysis are presented in Example 3.

[0202] 2.3.0 Production of Virus-Like Particles and Use of the Constructs of the Present Invention to Create Packaging Cell Lines.

[0203] The group-specific antigens (Gag) of human immunodeficiency virus type-1 (HIV-1) self-assemble into noninfectious virus-like particles (VLP) that are released from various eucaryotic cells by budding (reviewed by Freed, E. O., Virology 251:1-15, 1998). The Gag-containing synthetic expression cassettes of the present invention provide for the production of HIV-Gag virus-like particles (VLPs) using a variety of different cell types, including, but not limited to, mammalian cells.

[0204] Viral particles can be used as a matrix for the proper presentation of an antigen entrapped or associated therewith to the immune system of the host.

[0205] 2.3.1 VLP Production Using the Synthetic Expression Cassettes of the Present Invention

[0206] The Gag-containing synthetic expression cassettes of the present invention may provide superior production of both Gag proteins and VLPs, relative to native Gag coding sequences. Further, electron microscopic evaluation of VLP production can be used to show that free and budding immature virus particles of the expected size are produced by cells containing the synthetic expression cassettes.

[0207] Using the synthetic expression cassettes of the present invention, rather than native Gag coding sequences, for the production of virus-like particles provide several advantages. First, VLPs can be produced in enhanced quantity making isolation and purification of the VLPs easier. Second, VLPs can be produced in a variety of cell types using the synthetic expression cassettes, in particular, mammalian cell lines can be used for VLP production, for example, CHO cells. Production using CHO cells provides (i) VLP formation; (ii) correct myristoylation and budding; (iii) absence of non-Macmillian cell contaminants (e.g., insect viruses and/or cells); and (iv) ease of purification. The synthetic expression cassettes of the present invention are also useful for enhanced expression in cell-types other than mammalian cell lines. For example, infection of insect cells with baculovirus vectors encoding the synthetic expression cassettes results in higher levels of total Gag protein yield and higher levels of VLP production (relative to wild-coding sequences). Further, the final product from insect cells infected with the baculovirus-Gag synthetic expression cassettes consistently contains lower amounts of contaminating insect proteins than the final product when wild-coding sequences are used.

[0208] VLPs can spontaneously form when the particle-forming polypeptide of interest is recombinantly expressed in an appropriate host cell. Thus, the VLPs produced using the synthetic expression cassettes of the present invention are conveniently prepared using recombinant techniques. As discussed below, the Gag polypeptide encoding synthetic expression cassettes of the present invention can include other polypeptide coding sequences of interest (for example, HIV protease, HIV polymerase, Env; synthetic Env). Expression of such synthetic expression cassettes yields VLPs comprising the Gag polypeptide, as well as, the polypeptide of interest.

[0209] Once coding sequences for the desired particle-forming polypeptides have been isolated or synthesized, they can be cloned into any suitable vector or replicon for expression. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. See, generally, Sambrook et al, supra. The vector is then used to transform an appropriate host cell. Suitable recombinant expression systems include, but are not limited to, bacterial, mammalian, baculovirus/insect, vaccinia, Semliki Forest virus (SFV), Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis (VEE)), mammalian, yeast and Xenopus expression systems, well known in the art. Particularly preferred expression systems are mammalian cell lines, vaccinia, Sindbis, eucaryotic layered vector initiation systems (e.g., U.S. Pat. No. 6,015,686, U.S. Pat. No. 5,814,482, U.S. Pat. No. 6,015,694, U.S. Pat. No. 5,789,245, EP 1029068A2, WO 9918226A2/A3, EP 00907746A2, WO 9738087A2, all herein incorporated by reference in their entireties), insect and yeast systems.

[0210] The synthetic DNA fragments for the expression cassettes of the present invention, e.g., Pol, Gag, Env, Tat, Rev, Nef, Vif, Vpr, and/or Vpu, may be cloned into the following eucaryotic expression vectors: pCMVKm2, for transient expression assays and DNA immunization studies, the pCMVKm2 vector is derived from pCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) and comprises a kanamycin selectable marker, a ColE1 origin of replication, a CMV promoter enhancer and Intron A, followed by an insertion site for the synthetic sequences described below followed by a polyadenylation signal derived from bovine growth hormone--the pCMVKm2 vector differs from the pCMV-link vector only in that a polylinker site is inserted into pCMVKm2 to generate pCMV-link; pESN2dhfr and pCMVPLEdhfr, for expression in Chinese Hamster Ovary (CHO) cells; and, pAcC13, a shuttle vector for use in the Baculovirus expression system (pAcC13, is derived from pAcC12 which is described by Munemitsu S., et al., Mol Cell Biol. 10(11):5977-5982, 1990).

[0211] Briefly, construction of pCMVPLEdhfr was as follows.

[0212] To construct a DHFR cassette, the EMCV IRES (internal ribosome entry site) leader was PCR-amplified from pCite-4a+ (Novagen, Inc., Milwaukee, Wis.) and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as an Xba-Nco fragment to give pET-EMCV. The dhfr gene was PCR-amplified from pESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place of the translation stop codon and inserted as an Nco-BamH1 fragment to give pET-E-DHFR. Next, the attenuated neo gene was PCR amplified from a pSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into the unique BamH1 site of pET-E-DHFR to give pET-E-DHFR/Neo.sub.(m2). Finally the bovine growth hormone terminator from pCDNA3 (Invitrogen, Inc., Carlsbad, Calif.) was inserted downstream of the neo gene to give pET-E-DHFR/Neo.sub.(m2)BGHt. The EMCV-dhfr/neo selectable marker cassette fragment was prepared by cleavage of pET-E-DHFR/Neo.sub.(m2)BGHt.

[0213] In one vector construct the CMV enhancer/promoter plus Intron A was transferred from pCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-Sal1 fragment into pUC19 (New England Biolabs, Inc., Beverly, Mass.). The vector backbone of pUC19 was deleted from the Nde1 to the Sap1 sites. The above described DHFR cassette was added to the construct such that the EMCV IRES followed the CMV promoter. The vector also contained an amp.sup.r gene and an SV40 origin of replication.

[0214] A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (A.T.C.C.), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. See, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).

[0215] Viral vectors can be used for the production of particles in eucaryotic cells, such as those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. Additionally, a vaccinia based infection/transfection system, as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113, will also find use with the present invention. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. Alternately, T7 can be added as a purified protein or enzyme as in the "Progenitor" system (Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130). The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0216] Depending on the expression system and host selected, the VLPS are produced by growing host cells transformed by an expression vector under conditions whereby the particle-forming polypeptide is expressed and VLPs can be formed. The selection of the appropriate growth conditions is within the skill of the art. If the VLPs are formed intracellularly, the cells are then disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the VLPs substantially intact. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).

[0217] The particles are then isolated (or substantially purified) using methods that preserve the integrity thereof, such as, by gradient centrifugation, e.g., cesium chloride (CsCl) sucrose gradients, pelleting and the like (see, e.g., Kimbauer et al. J. Virol. (1993) 67:6929-6936), as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography.

[0218] VLPs produced by cells containing the synthetic expression cassettes of the present invention can be used to elicit an immune response when administered to a subject. One advantage of the present invention is that VLPs can be produced by mammalian cells carrying the synthetic expression cassettes at levels previously not possible. As discussed above, the VLPs can comprise a variety of antigens in addition to the Gag polypeptide (e.g., Gag-protease, Gag-polymerase, Env, synthetic Env, etc.). Purified VLPs, produced using the synthetic expression cassettes of the present invention, can be administered to a vertebrate subject, usually in the form of vaccine compositions. Combination vaccines may also be used, where such vaccines contain, for example, an adjuvant subunit protein (e.g., Env). Administration can take place using the VLPs formulated alone or formulated with other antigens. Further, the VLPs can be administered prior to, concurrent with, or subsequent to, delivery of the synthetic expression cassettes for DNA immunization (see below) and/or delivery of other vaccines. Also, the site of VLP administration may be the same or different as other vaccine compositions that are being administered. Gene delivery can be accomplished by a number of methods including, but are not limited to, immunization with DNA, alphavirus vectors, pox virus vectors, and vaccinia virus vectors.

[0219] VLP immune-stimulating (or vaccine) compositions can include various excipients, adjuvants, carriers, auxiliary substances, modulating agents, and the like. The immune stimulating compositions will include an amount of the VLP/antigen sufficient to mount an immunological response. An appropriate effective amount can be determined by one of skill in the art. Such an amount will fall in a relatively broad range that can be determined through routine trials and will generally be an amount on the order of about 0.1 .mu.g to about 1000 .mu.g, more preferably about 1 .mu.g to about 300 .mu.g, of VLP/antigen.

[0220] A carrier is optionally present which is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul. 14(2): 197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen may be conjugated to a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc., as well as toxins derived from E. coli.

[0221] Adjuvants may also be used to enhance the effectiveness of the compositions. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (3) saponin adjuvants, such as Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) oligonucleotides or polymeric molecules encoding immunostimulatory CpG motifs (Davis, H. L., et al., J. Immunology 160:870-876, 1998; Sato, Y. et al., Science 273:352-354, 1996) or complexes of antigens/oligonucleotides (Polymeric molecules include double and single stranded RNA and DNA, and backbone modifications thereof, for example, methylphosphonate linkages; or (7) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. W093/13202 and W092/19265); and (8) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Further, such polymeric molecules include alternative polymer backbone structures such as, but not limited to, polyvinyl backbones (Pitha, Biochem Biophys Acta, 204:39, 1970a; Pitha, Biopolymers, 9:965, 1970b), and morpholino backbones (Summerton, J., et al., U.S. Pat. No. 5,142,047, issued Aug. 25, 1992; Summerton, J., et al., U.S. Pat. No. 5,185,444 issued Feb. 9, 1993). A variety of other charged and uncharged polynucleotide analogs have been reported. Numerous backbone modifications are known in the art, including, but not limited to, uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, and carbamates) and charged linkages (e.g., phosphorothioates and phosphorodithioates).}; and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the VLP immune-stimulating (or vaccine) composition. Alum, CpG oligonucleotides, and MF59 are preferred.

[0222] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-s- n-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

[0223] Dosage treatment with the VLP composition may be a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals, chosen to maintain and/or reinforce the immune response, for example at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgment of the practitioner.

[0224] If prevention of disease is desired, the antigen carrying VLPs are generally administered prior to primary infection with the pathogen of interest. If treatment is desired, e.g., the reduction of symptoms or recurrences, the VLP compositions are generally administered subsequent to primary infection.

[0225] 2.3.2 Using the Synthetic Expression Cassettes of the Present Invention to Create Packaging Cell Lines

[0226] A number of viral based systems have been developed for use as gene transfer vectors for mammalian host cells. For example, retroviruses (in particular, antiviral vectors) provide a convenient platform for gene delivery systems. A coding sequence of interest (for example, a sequence useful for gene therapy applications) can be inserted into a gene delivery vector and packaged in retroviral particles using techniques known in the art. Recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described, including, for example, the following: (U.S. Pat. No. 5,219,740; Miller et al. (1989) BioTechniques 7:980; Miller, A. D. (1990) Human Gene Therapy 1:5; Scarpa et al. (1991) Virology 180:849; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033; Boris-Lawrie et al. (1993) Cur. Opin. Genet. Develop. 3:102; GB 2200651; EP 0415731; EP 0345242; WO 89/02468; WO 89/05349; WO 89/09271; WO 90/02806; WO 90/07936; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; in U.S. Pat. No. 5,219,740; U.S. Pat. No. 4,405,712; U.S. Pat. No. 4,861,719; U.S. Pat. No. 4,980,289 and U.S. Pat. No. 4,777,127; in U.S. Ser. No. 07/800,921; and in Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53:83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci USA 81; 6349; and Miller (1990) Human Gene Therapy 1.

[0227] In other embodiments, gene transfer vectors can be constructed to encode a cytokine or other immunomodulatory molecule. For example, nucleic acid sequences encoding native IL-2 and gamma-interferon can be obtained as described in U.S. Pat. Nos. 4,738,927 and 5,326,859, respectively, while useful muteins of these proteins can be obtained as described in U.S. Pat. No. 4,853,332. Nucleic acid sequences encoding the short and long forms of mCSF can be obtained as described in U.S. Pat. Nos. 4,847,201 and 4,879,227, respectively. In particular aspects of the invention, retroviral vectors expressing cytokine or immunomodulatory genes can be produced as described herein (for example, employing the packaging cell lines of the present invention) and in International Application No. PCT US 94/02951, entitled "Compositions and Methods for Cancer Immunotherapy."

[0228] Examples of suitable immunomodulatory molecules for use herein include the following: IL-1 and IL-2 (Karupiah et al. (1990) J. Immunology 144:290-298, Weber et al. (1987) J. Exp. Med. 166:1716-1733, Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224, and U.S. Pat. No. 4,738,927); IL-3 and IL-4 (Tepper et al. (1989) Cell 57:503-512, Golumbek et al. (1991) Science 254:713-716, and U.S. Pat. No. 5,017,691); IL-5 and IL-6 (Brakenhof et al. (1987) J. Immunol. 139:4116-4121, and International Publication No. WO 90/06370); IL-7 (U.S. Pat. No. 4,965,195); IL-8, IL-9, IL-10, IL-11, IL-12, and IL-13 (Cytokine Bulletin, Summer 1994); IL-14 and IL-15; alpha interferon (Finter et al. (1991) Drugs 42:749-765, U.S. Pat. Nos. 4,892,743 and 4,966,843, International Publication No. WO 85/02862, Nagata et al. (1980) Nature 284:316-320, Familletti et al. (1981) Methods in Enz. 78:387-394, Twu et al. (1989) Proc. Natl. Acad. Sci. USA 86:2046-2050, and Faktor et al. (1990) Oncogene 5:867-872); beta-interferon (Seif et al. (1991) J. Virol. 65:664-671); gamma-interferons (Radford et al. (1991) The American Society of Hepatology 20082015, Watanabe et al. (1989) Proc. Natl. Acad. Sci. USA 86:9456-9460, Gansbacher et al. (1990) Cancer Research 50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother. 30:34-42, and U.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat. Nos. 4,999,291 and 4,810,643); GM-CSF (International Publication No. WO 85/04188).

[0229] Immunomodulatory factors may also be agonists, antagonists, or ligands for these molecules. For example, soluble forms of receptors can often behave as antagonists for these types of factors, as can mutated forms of the factors themselves.

[0230] Nucleic acid molecules that encode the above-described substances, as well as other nucleic acid molecules that are advantageous for use within the present invention, may be readily obtained from a variety of sources, including, for example, depositories such as the American Type Culture Collection, or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford England). Representative examples include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which contains sequences encoding gamma interferon), A.T.C.C. Deposit No. 39656 (which contains sequences encoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequences encoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517 (which contain sequences encoding beta-interferon), A.T.C.C. Deposit No. 67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C. Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which contain sequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398, and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C. Deposit No. 57592 (which contains sequences encoding Interleukin-4), A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encoding Interleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequences encoding Interleukin-6).

[0231] Plasmids containing cytokine genes or immunomodulatory genes (International Publication Nos. WO 94/02951 and WO 96/21015, both of which are incorporated by reference in their entirety) can be digested with appropriate restriction enzymes, and DNA fragments containing the particular gene of interest can be inserted into a gene transfer vector using standard molecular biology techniques. (See, e.g., Sambrook et al., supra., or Ausubel et al. (eds) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience).

[0232] Polynucleotide sequences coding for the above-described molecules can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from a vector known to include the same. For example, plasmids which contain sequences that encode altered cellular products may be obtained from a depository such as the A.T.C.C., or from commercial sources. Plasmids containing the nucleotide sequences of interest can be digested with appropriate restriction enzymes, and DNA fragments containing the nucleotide sequences can be inserted into a gene transfer vector using standard molecular biology techniques.

[0233] Alternatively, cDNA sequences for use with the present invention may be obtained from cells which express or contain the sequences, using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA. Briefly, mRNA from a cell which expresses the gene of interest can be reverse transcribed with reverse transcriptase using oligo-dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159, see also PCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989)) using oligonucleotide primers complementary to sequences on either side of desired sequences.

[0234] The nucleotide sequence of interest can also be produced synthetically, rather than cloned, using a DNA synthesizer (e.g., an Applied Biosystems Model 392 DNA Synthesizer, available from ABI, Foster City, Calif.). The nucleotide sequence can be designed with the appropriate codons for the expression product desired. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.

[0235] The synthetic expression cassettes of the present invention can be employed in the construction of packaging cell lines for use with retroviral vectors.

[0236] One type of retrovirus, the murine leukemia virus, or "MLV", has been widely utilized for gene therapy applications (see generally Mann et al. (Cell 33:153, 1993), Cane and Mulligan (Proc, Nat'l. Acad. Sci. USA 81:6349, 1984), and Miller et al., Human Gene Therapy 1:5-14, 1990.

[0237] Lentiviral vectors typically, comprise a 5' lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to one or more genes of interest, an origin of second strand DNA synthesis and a 3' lentiviral LTR, wherein the lentiviral vector contains a nuclear transport element. The nuclear transport element may be located either upstream (5') or downstream (3') of a coding sequence of interest (for example, a synthetic Gag or Env expression cassette of the present invention). Within certain embodiments, the nuclear transport element is not RRE. Within one embodiment the packaging signal is an extended packaging signal. Within other embodiments the promoter is a tissue specific promoter, or, alternatively, a promoter such as CMV. Within other embodiments, the lentiviral vector further comprises an internal ribosome entry site.

[0238] A wide variety of lentiviruses may be utilized within the context of the present invention, including for example, lentiviruses selected from the group consisting of HIV, HIV-1, HIV-2, FIV and SIV.

[0239] Within yet another aspect of the invention, host cells (e.g., packaging cell lines) are provided which contain any of the expression cassettes described herein. For example, within one aspect packaging cell line are provided comprising an expression cassette that comprises a sequence encoding synthetic Gag-polymerase, and a nuclear transport element, wherein the promoter is operably linked to the sequence encoding Gag-polymerase. Packaging cell lines may further comprise a promoter and a sequence encoding tat, rev, or an envelope, wherein the promoter is operably linked to the sequence encoding tat, rev, Env or sequences encoding modified versions of these proteins. The packaging cell line may further comprise a sequence encoding any one or more of other HIV gene encoding sequences.

[0240] In one embodiment, the expression cassette (carrying, for example, the synthetic Gag-polymerase) is stably integrated. The packaging cell line, upon introduction of a lentiviral vector, typically produces particles. The promoter regulating expression of the synthetic expression cassette may be inducible. Typically, the packaging cell line, upon introduction of a lentiviral vector, produces particles that are essentially free of replication competent virus.

[0241] Packaging cell lines are provided comprising an expression cassette which directs the expression of a synthetic Gag-polymerase gene or comprising an expression cassette which directs the expression of a synthetic Env genes described herein. (See, also, Andre, S., et al., Journal of Virology 72(2):1497-1503, 1998; Haas, J., et al., Current Biology 6(3):315-324, 1996) for a description of other modified Env sequences). A lentiviral vector is introduced into the packaging cell line to produce a vector producing cell line.

[0242] As noted above, lentiviral vectors can be designed to carry or express a selected gene(s) or sequences of interest. Lentiviral vectors may be readily constructed from a wide variety of lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Representative examples of lentiviruses included HIV, HIV-1, HIV-2, FIV and SIV. Such lentiviruses may either be obtained from patient isolates, or, more preferably, from depositories or collections such as the American Type Culture Collection, or isolated from known sources using available techniques.

[0243] Portions of the lentiviral gene delivery vectors (or vehicles) may be derived from different viruses. For example, in a given recombinant lentiviral vector, LTRs may be derived from an HIV, a packaging signal from SIV, and an origin of second strand synthesis from HrV-2. Lentiviral vector constructs may comprise a 5' lentiviral LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR, wherein said lentiviral vector contains a nuclear transport element that is not RRE.

[0244] Briefly, Long Terminal Repeats ("LTRs") are subdivided into three elements, designated U5, R and U3. These elements contain a variety of signals which are responsible for the biological activity of a retrovirus, including for example, promoter and enhancer elements which are located within U3. LTRs may be readily identified in the provirus (integrated DNA form) due to their precise duplication at either end of the genome. As utilized herein, a 5' LTR should be understood to include a 5' promoter element and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector. The 3' LTR should be understood to include a polyadenylation signal, and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.

[0245] The tRNA binding site and origin of second strand DNA synthesis are also important for a retrovirus to be biologically active, and may be readily identified by one of skill in the art. For example, retroviral tRNA binds to a tRNA binding site by Watson-Crick base pairing, and is carried with the retrovirus genome into a viral particle. The tRNA is then utilized as a primer for DNA synthesis by reverse transcriptase. The tRNA binding site may be readily identified based upon its location just downstream from the 5'LTR. Similarly, the origin of second strand DNA synthesis is, as its name implies, important for the second strand DNA synthesis of a retrovirus. This region, which is also referred to as the poly-purine tract, is located just upstream of the 3'LTR.

[0246] In addition to a 5' and 3' LTR, tRNA binding site, and origin of second strand DNA synthesis, recombinant retroviral vector constructs may also comprise a packaging signal, as well as one or more genes or coding sequences of interest. In addition, the lentiviral vectors have a nuclear transport element which, in preferred embodiments is not RRE. Representative examples of suitable nuclear transport elements include the element in Rous sarcoma virus (Ogert, et al., J ViroL 70, 3834-3843, 1996), the element in Rous sarcoma virus (Liu & Mertz, Genes & Dev., 9, 1766-1789, 1995) and the element in the genome of simian retrovirus type I (Zolotukhin, et al., J Virol. 68, 7944-7952, 1994). Other potential elements include the elements in the histone gene (Kedes, Annu. Rev. Biochem. 48, 837-870, 1970), the .alpha.-interferon gene (Nagata et al., Nature 287, 401-408, 1980), the .beta.-adrenergic receptor gene (Koilka, et al., Nature 329, 75-79, 1987), and the c-Jun gene (Hattorie, et al., Proc. Natl. Acad. Sci. USA 85, 9148-9152, 1988).

[0247] Recombinant lentiviral vector constructs typically lack both Gag-polymerase and Env coding sequences. Recombinant lentiviral vector typically contain less than 20, preferably 15, more preferably 10, and most preferably 8 consecutive nucleotides found in Gag-polymerase and Env genes. One advantage of the present invention is that the synthetic Gag-polymerase expression cassettes, which can be used to construct packaging cell lines for the recombinant retroviral vector constructs, have little homology to wild-type Gag-polymerase sequences and thus considerably reduce or eliminate the possibility of homologous recombination between the synthetic and wild-type sequences.

[0248] Lentiviral vectors may also include tissue-specific promoters to drive expression of one or more genes or sequences of interest.

[0249] Lentiviral vector constructs may be generated such that more than one gene of interest is expressed. This may be accomplished through the use of di- or oligo-cistronic cassettes (e.g., where the coding regions are separated by 80 nucleotides or less, see generally Levin et al., Gene 108:167-174, 1991), or through the use of Internal Ribosome Entry Sites ("IRES").

[0250] Packaging cell lines suitable for use with the above described recombinant retroviral vector constructs may be readily prepared given the disclosure provided herein. Briefly, the parent cell line from which the packaging cell line is derived can be selected from a variety of mammalian cell lines, including for example, 293, RD, COS-7, CHO, BHK, VERO, HT1080, and myeloma cells.

[0251] After selection of a suitable host cell for the generation of a packaging cell line, one or more expression cassettes are introduced into the cell line in order to complement or supply in trans components of the vector which have been deleted.

[0252] Representative examples of suitable synthetic HIV polynucleotide sequences have been described herein for use in expression cassettes of the present invention. As described above, the native and/or synthetic coding sequences may also be utilized in these expression cassettes.

[0253] Utilizing the above-described expression cassettes, a wide variety of packaging cell lines can be generated. For example, within one aspect packaging cell line are provided comprising an expression cassette that comprises a sequence encoding synthetic Gag-polymerase, and a nuclear transport element, wherein the promoter is operably linked to the sequence encoding Gag-polymerase. Within other aspects, packaging cell lines are provided comprising a promoter and a sequence encoding tat, rev, Env, or other HIV antigens or epitopes derived therefrom, wherein the promoter is operably linked to the sequence encoding tat, rev, Env, or the HIV antigen or epitope. Within further embodiments, the packaging cell line may comprise a sequence encoding any one or more of tat, rev, nef, vif, vpu or vpr. For example, the packaging cell line may contain only tat, rev, nef, vif, vpu, or vpr alone, tat rev and nef, nef and vif, nef and vpu, nef and vpr, vif and vpu, vif and vpr, vpu and vpr, nef vif and vpu, nef vif and vpr, nef vpu and vpr, vif vpu and vpr, all four of nef, vif, vpu, and vpr, etc.

[0254] In one embodiment, the expression cassette is stably integrated. Within another embodiment, the packaging cell line, upon introduction of a lentiviral vector, produces particles. Within further embodiments the promoter is inducible. Within certain preferred embodiments of the invention, the packaging cell line, upon introduction of a lentiviral vector, produces particles that are free of replication competent virus.

[0255] The synthetic cassettes containing modified coding sequences are transfected into a selected cell line. Transfected cells are selected that (i) carry, typically, integrated, stable copies of the HIV coding sequences, and (ii) are expressing acceptable levels of these polypeptides (expression can be evaluated by methods known in the prior art in view of the teachings of the present disclosure). The ability of the cell line to produce VLPs may also be verified.

[0256] A sequence of interest is constructed into a suitable viral vector as discussed above. This defective virus is then transfected into the packaging cell line. The packaging cell line provides the viral functions necessary for producing virus-like particles into which the defective viral genome, containing the sequence of interest, are packaged. These VLPs are then isolated and can be used, for example, in gene delivery or gene therapy.

[0257] Further, such packaging cell lines can also be used to produce VLPs alone, which can, for example, be used as adjuvants for administration with other antigens or in vaccine compositions. Also, co-expression of a selected sequence of interest encoding a polypeptide (for example, an antigen) in the packaging cell line can also result in the entrapment and/or association of the selected polypeptide in/with the VLPs.

[0258] Various forms of the different embodiments of the present invention (e.g., synthetic constructs) may be combined.

[0259] 2.4.0 DNA Immunization and Gene Delivery

[0260] A variety of HIV polypeptide antigens, particularly HIV antigens, can be used in the practice of the present invention. HIV antigens can be included in DNA immunization constructs containing, for example, a synthetic Env expression cassettes, a synthetic Gag expression cassette, a synthetic pol-derived polypeptide expression cassette, a synthetic expression cassette comprising sequences encoding one or more accessory or regulatory genes (e.g., tat, rev, nef, vif, vpu, vpr), and/or a synthetic Gag expression cassette fused in-frame to a coding sequence for the polypeptide antigen (synthetic or wild-type), where expression of the construct results in VLPs presenting the antigen of interest.

[0261] HIV antigens of particular interest to be used in the practice of the present invention include pol, tat, rev, nef, vif, vpu, vpr, and other HIV-1 (also known as HTLV-III, LAV, ARV, etc.) antigens or epitopes derived therefrom, including, but not limited to, antigens such as gp120, gp41, gp160 (both native and modified); Gag; and pol from a variety of isolates including, but not limited to, HIV.sub.IIIb, HIV.sub.SF2, HIV-1.sub.SF162, HIV-1.sub.SF170, HIV.sub.LAV, HIV.sub.LAI, HIV.sub.MN, HIV-1.sub.CM235, HIV-1.sub.US4, other HIV-1 strains from diverse subtypes (e.g., subtypes, A through G, and O), HIV-2 strains and diverse subtypes (e.g., HIV-2.sub.UC1 and HIV-2.sub.UC2). See, e.g., Myers, et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N. Mex.; Myers, et al., Human Retroviruses and Aids, 1990, Los Alamos, N. Mex.: Los Alamos National Laboratory. These antigens may be synthetic (as described herein) or wild-type.

[0262] To evaluate efficacy, DNA immunization using synthetic expression cassettes of the present invention can be performed, for example, as follows. Mice are immunized with a tat/rev/nef synthetic expression cassette. Other mice are immunized with a tat/rev/nef wild type expression cassette. Mouse immunizations with plasmid-DNAs typically show that the synthetic expression cassettes provide a clear improvement of immunogenicity relative to the native expression cassettes. Also, a second boost immunization will induce a secondary immune response, for example, after approximately two weeks. Further, the results of CTL assays typically show increased potency of synthetic expression cassettes for induction of cytotoxic T-lymphocyte (CTL) responses by DNA immunization.

[0263] Exemplary primate studies directed at the evaluation of neutralizing antibodies and cellular immune responses against HIV are described below.

[0264] It is readily apparent that the subject invention can be used to mount an immune response to a wide variety of antigens and hence to treat or prevent infection, particularly HIV infection.

[0265] 2.4.1 Delivery of the Synthetic Expression Cassettes of the Present Invention

[0266] Polynucleotide sequences coding for the above-described molecules can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from a vector known to include the same. Furthermore, the desired gene can be isolated directly from cells and tissues containing the same, using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA. The gene of interest can also be produced synthetically, rather than cloned. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311; Stemmer, W. P. C., (1995) Gene 164:49-53.

[0267] Next, the gene sequence encoding the desired antigen can be inserted into a vector containing a synthetic expression cassette of the present invention. In one embodiment, polynucleotides encoding selected antigens are separately cloned into expression vectors (e.g., Env-coding polynucleotide in a first vector, Gag-coding polynucleotide in a second vector, Pol-derived polypeptide-coding polynucleotide in a third vector, tat-, rev-, nef-, vif-, vpu-, vpr-coding polynucleotides in further vectors, etc.). In certain embodiments, the antigen is inserted into or adjacent a synthetic Gag coding sequence such that when the combined sequence is expressed it results in the production of VLPs comprising the Gag polypeptide and the antigen of interest, e.g., Env (native or modified) or other antigen(s) (native or modified) derived from HIV. Insertions can be made within the coding sequence or at either end of the coding sequence (5', amino terminus of the expressed Gag polypeptide; or 3', carboxy terminus of the expressed Gag polypeptide)(Wagner, R., et al., Arch Virol. 127:117-137, 1992; Wagner, R., et al., Virology 200:162-175, 1994; Wu, X., et al., J. Virol. 69(6):3389-3398, 1995; Wang, C-T., et al., Virology 200:524-534, 1994; Chazal, N., et al., Virology 68(1):111-122, 1994; Griffiths, J. C., et al., J. Virol. 67(6):3191-3198, 1993; Reicin, A. S., et al., J. Virol. 69(2):642-650, 1995).

[0268] Up to 50% of the coding sequences of p55Gag can be deleted without affecting the assembly to virus-like particles and expression efficiency (Borsetti, A., et al, J. Virol. 72(11):9313-9317, 1998; Gamier, L., et al., J Virol 72(6):4667-4677, 1998; Zhang, Y., et al., J Virol 72(3): 1782-1789, 1998; Wang, C., et al., J Virol 72(10): 7950-7959, 1998). In one embodiment of the present invention, immunogenicity of the high level expressing synthetic Gag expression cassettes can be increased by the insertion of different structural or non-structural HIV antigens, multi-epitope cassettes, or cytokine sequences into deleted regions of Gag sequence. Such deletions may be generated following the teachings of the present invention and information available to one of ordinary skill in the art. One possible advantage of this approach, relative to using full-length sequences fused to heterologous polypeptides, can be higher expression/secretion efficiency of the expression product.

[0269] When sequences are added to the amino terminal end of Gag, the polynucleotide can contain coding sequences at the 5' end that encode a signal for addition of a myristic moiety to the Gag-containing polypeptide (e.g., sequences that encode Met-Gly).

[0270] The ability of Gag-containing polypeptide constructs to form VLPs can be empirically determined following the teachings of the present specification.

[0271] The synthetic expression cassettes can also include control elements operably linked to the coding sequence, which allow for the expression of the gene in vivo in the subject species. For example, typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0272] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.

[0273] Furthermore, plasmids can be constructed which include a chimeric antigen-coding gene sequences, encoding, e.g., multiple antigens/epitopes of interest, for example derived from more than one viral isolate.

[0274] Typically the antigen coding sequences precede or follow the synthetic coding sequence and the chimeric transcription unit will have a single open reading frame encoding both the antigen of interest and the synthetic coding sequences. Alternatively, multi-cistronic cassettes (e.g., bi-cistronic cassettes) can be constructed allowing expression of multiple antigens from a single mRNA using the EMCV IRES, or the like (Example 7).

[0275] In one embodiment of the present invention, a nucleic acid immunizing composition may comprise, for example, the following: a first expression vector comprising a Gag expression cassette, a second vector comprising an Env expression cassette, and a third expression vector comprising a Pol expression cassette, or one or more coding region of Pol (e.g., Prot, RT, RNase, Int), wherein further antigen coding sequences may be associated with the Pol expression, such antigens may be obtained, for example, from accessory genes (e.g., vpr, vpu, vif), regulatory genes (e.g., nef, tat, rev), or portions of the Pol sequences (e.g., Prot, RT, RNase, Int)). In another embodiment, a nucleic acid immunizing composition may comprise, for example, an expression cassette comprising any of the synthetic polynucleotide sequences of the present invention. In another embodiment, a nucleic acid immunizing composition may comprise, for example, an expression cassette comprising coding sequences for a number of HIV genes (or sequences derived from such genes) wherein the coding sequences are in-frame and under the control of a single promoter, for example, Gag-Env constructs, Tat-Rev-Nef constructs, P2Pol-tat-rev-nef constructs, etc. The synthetic coding sequences of the present invention may be combined in any number of combinations depending on the coding sequence products (i.e., HIV polypeptides) to which, for example, an immunological response is desired to be raised. In yet another embodiment, synthetic coding sequences for multiple HIV-derived polypeptides may be constructed into a polycistronic message under the control of a single promoter wherein IRES are placed adjacent the coding sequence for each encoded polypeptide.

[0276] Once complete, the constructs are used for nucleic acid immunization using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Genes can be delivered either directly to the vertebrate subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.

[0277] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

[0278] A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

[0279] Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

[0280] Another vector system useful for delivering the polynucleotides of the present invention is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

[0281] Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the antigens of interest include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the genes can be constructed as follows. The DNA encoding the particular synthetic HIV polypeptide coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK.sup.- recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0282] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0283] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0284] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis, Semliki Forest, and Venezuelan Equine Encephalitis viruses, will also find use as viral vectors for delivering the polynucleotides of the present invention (for example, a synthetic Gag-polypeptide encoding expression cassette). For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Preferred expression systems include, but are not limited to, eucaryotic layered vector initiation systems (e.g., U.S. Pat. No. 6,015,686, U.S. Pat. No. 5,814,482, U.S. Pat. No. 6,015,694, U.S. Pat. No. 5,789,245, EP 1029068A2, WO 9918226A2/A3, EP 00907746A2, WO 9738087A2, all herein incorporated by reference in their entireties).

[0285] A vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0286] As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.

[0287] Delivery of the expression cassettes of the present invention can also be accomplished using eucaryotic expression vectors comprising CMV-derived elements, such vectors include, but are not limited to, the following: pCMVKm2, pCMV-link pCMVPLEdhfr, and pCMV6a (all described above).

[0288] Synthetic expression cassettes of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.

[0289] Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.

[0290] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No. WO 90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0291] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0292] The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0293] The DNA and/or protein antigen(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

[0294] The synthetic expression cassette of interest may also be encapsulated; adsorbed to, or associated with, particulate carriers. Such carriers present multiple copies of a selected antigen to the immune system and promote trapping and retention of antigens in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993. Suitable microparticles may also be manufactured in the presence of charged detergents, such as anionic or cationic detergents, to yield microparticles with a surface having a net negative or a net positive charge. For example, microparticles manufactured with anionic detergents, such as hexadecyltrimethylammonium bromide (CTAB), i.e. CTAB-PLG microparticles, adsorb negatively charged macromolecules, such as DNA. (see, e.g., Int'l Application Number PCT/US99/17308).

[0295] Furthermore, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the gene of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Felgner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No. 5,831,005, issued Nov. 3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present invention.

[0296] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes of the present invention. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0297] Recombinant vectors carrying a synthetic expression cassette of the present invention are formulated into compositions for delivery to the vertebrate subject. These compositions may either be prophylactic (to prevent infection) or therapeutic (to treat disease after infection). The compositions will comprise a "therapeutically effective amount" of the gene of interest such that an amount of the antigen can be produced in vivo so that an immune response is generated in the individual to which it is administered. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the capacity of the subject's immune system to synthesize antibodies; the degree of protection desired; the severity of the condition being treated; the particular antigen selected and its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials.

[0298] The compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Certain facilitators of nucleic acid uptake and/or expression can also be included in the compositions or coadministered, such as, but not limited to, bupivacaine, cardiotoxin and sucrose.

[0299] Once formulated, the compositions of the invention can be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) (with or without the corresponding antigen) in liposomes, and direct microinjection of the DNA into nuclei.

[0300] Direct delivery of synthetic expression cassette compositions in vivo will generally be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe or a gene gun, such as the Accell.RTM. gene delivery system (PowderJect Technologies, Inc., Oxford, England). The constructs can be injected either subcutaneously, epidermally, intradermally, intramucosally such as nasally, rectally and vaginally, intraperitoneally, intravenously, orally or intramuscularly. Delivery of DNA into cells of the epidermis is particularly preferred as this mode of administration provides access to skin-associated lymphoid cells and provides for a transient presence of DNA in the recipient. Other modes of administration include oral and pulmonary administration, suppositories, needle-less injection, transcutaneous and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. Administration of nucleic acids may also be combined with administration of peptides or other substances.

[0301] Exemplary immunogenicity studies are presented in Examples 4, 5, 6, 9, 10, 11, and 12.

[0302] 2.4.2 Ex Vivo Delivery of the Synthetic Expression Cassettes of the Present Invention

[0303] In one embodiment, T cells, and related cell types (including but not limited to antigen presenting cells, such as, macrophage, monocytes, lymphoid cells, dendritic cells, B-cells, T-cells, stem cells, and progenitor cells thereof), can be used for ex vivo delivery of the synthetic expression cassettes of the present invention. T cells can be isolated from peripheral blood lymphocytes (PBLs) by a variety of procedures known to those skilled in the art. For example, T cell populations can be "enriched" from a population of PBLs through the removal of accessory and B cells. In particular, T cell enrichment can be accomplished by the elimination of non-T cells using anti-MHC class II monoclonal antibodies. Similarly, other antibodies can be used to deplete specific populations of non-T cells. For example, anti-Ig antibody molecules can be used to deplete B cells and anti-MacI antibody molecules can be used to deplete macrophages.

[0304] T cells can be further fractionated into a number of different subpopulations by techniques known to those skilled in the art. Two major subpopulations can be isolated based on their differential expression of the cell surface markers CD4 and CD8. For example, following the enrichment of T cells as described above, CD4.sup.+ cells can be enriched using antibodies specific for CD4 (see Coligan et al., supra). The antibodies may be coupled to a solid support such as magnetic beads. Conversely, CD8+ cells can be enriched through the use of antibodies specific for CD4 (to remove CD4.sup.+ cells), or can be isolated by the use of CD8 antibodies coupled to a solid support. CD4 lymphocytes from HIV-1 infected patients can be expanded ex vivo, before or after transduction as described by Wilson et. al. (1995) J. Infect. Dis. 172:88.

[0305] Following purification of T cells, a variety of methods of genetic modification known to those skilled in the art can be performed using non-viral or viral-based gene transfer vectors constructed as described herein. For example, one such approach involves transduction of the purified T cell population with vector-containing supernatant of cultures derived from vector producing cells. A second approach involves co-cultivation of an irradiated monolayer of vector-producing cells with the purified T cells. A third approach involves a similar co-cultivation approach; however, the purified T cells are pre-stimulated with various cytokines and cultured 48 hours prior to the co-cultivation with the irradiated vector producing cells. Pre-stimulation prior to such transduction increases effective gene transfer (Nolta et al. (1992) Exp. Hematol. 20:1065). Stimulation of these cultures to proliferate also provides increased cell populations for re-infusion into the patient. Subsequent to co-cultivation, T cells are collected from the vector producing cell monolayer, expanded, and frozen in liquid nitrogen.

[0306] Gene transfer vectors, containing one or more synthetic expression cassette of the present invention (associated with appropriate control elements for delivery to the isolated T cells) can be assembled using known methods and following the guidance of the present specification.

[0307] Selectable markers can also be used in the construction of gene transfer vectors. For example, a marker can be used which imparts to a mammalian cell transduced with the gene transfer vector resistance to a cytotoxic agent. The cytotoxic agent can be, but is not limited to, neomycin, aminoglycoside, tetracycline, chloramphenicol, sulfonamide, actinomycin, netropsin, distamycin A, anthracycline, or pyrazinamide. For example, neomycin phosphotransferase II imparts resistance to the neomycin analogue geneticin (G418).

[0308] The T cells can also be maintained in a medium containing at least one type of growth factor prior to being selected. A variety of growth factors are known in the art which sustain the growth of a particular cell type. Examples of such growth factors are cytokine mitogens such as rIL-2, IL-10, IL-12, and IL-15, which promote growth and activation of lymphocytes. Certain types of cells are stimulated by other growth factors such as hormones, including human chorionic gonadotropin (hCG) and human growth hormone. The selection of an appropriate growth factor for a particular cell population is readily accomplished by one of skill in the art.

[0309] For example, white blood cells such as differentiated progenitor and stem cells are stimulated by a variety of growth factors. More particularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF, produced by activated T.sub.H and activated macrophages, stimulate myeloid stem cells, which then differentiate into pluripotent stem cells, granulocyte-monocyte progenitors, eosinophil progenitors, basophil progenitors, megakaryocytes, and erythroid progenitors. Differentiation is modulated by growth factors such as GM-CSF, IL-3, IL-6, IL-11, and EPO.

[0310] Pluripotent stem cells then differentiate into lymphoid stem cells, bone marrow stromal cells, T cell progenitors, B cell progenitors, thymocytes, T.sub.H Cells, T.sub.C cells, and B cells. This differentiation is modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF, M-CSF, G-CSF, IL-2, and IL-5.

[0311] Granulocyte-monocyte progenitors differentiate to monocytes, macrophages, and neutrophils. Such differentiation is modulated by the growth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitors differentiate into eosinophils. This process is modulated by GM-CSF and IL-5.

[0312] The differentiation of basophil progenitors into mast cells and basophils is modulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produce platelets in response to GM-CSF, EPO, and IL-6. Erythroid progenitor cells differentiate into red blood cells in response to EPO.

[0313] Thus, during activation by the CD3-binding agent, T cells can also be contacted with a mitogen, for example a cytokine such as IL-2. In particularly preferred embodiments, the IL-2 is added to the population of T cells at a concentration of about 50 to 100 .mu.g/ml. Activation with the CD3-binding agent can be carried out for 2 to 4 days.

[0314] Once suitably activated, the T cells are genetically modified by contacting the same with a suitable gene transfer vector under conditions that allow for transfection of the vectors into the T cells. Genetic modification is carried out when the cell density of the T cell population is between about 0.1.times.10.sup.6 and 5.times.10.sup.6, preferably between about 0.5.times.10.sup.6 and 2.times.10.sup.6. A number of suitable viral and nonviral-based gene transfer vectors have been described for use herein.

[0315] After transduction, transduced cells are selected away from non-transduced cells using known techniques. For example, if the gene transfer vector used in the transduction includes a selectable marker which confers resistance to a cytotoxic agent, the cells can be contacted with the appropriate cytotoxic agent, whereby non-transduced cells can be negatively selected away from the transduced cells. If the selectable marker is a cell surface marker, the cells can be contacted with a binding agent specific for the particular cell surface marker, whereby the transduced cells can be positively selected away from the population. The selection step can also entail fluorescence-activated cell sorting (FACS) techniques, such as where FACS is used to select cells from the population containing a particular surface marker, or the selection step can entail the use of magnetically responsive particles as retrievable supports for target cell capture and/or background removal.

[0316] More particularly, positive selection of the transduced cells can be performed using a FACS cell sorter (e.g. a FACSVantage.TM. Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to sort and collect transduced cells expressing a selectable cell surface marker. Following transduction, the cells are stained with fluorescent-labeled antibody molecules directed against the particular cell surface marker. The amount of bound antibody on each cell can be measured by passing droplets containing the cells through the cell sorter. By imparting an electromagnetic charge to droplets containing the stained cells, the transduced cells can be separated from other cells. The positively selected cells are then harvested in sterile collection vessels. These cell sorting procedures are described in detail, for example, in the FACSVantage.TM. Training Manual, with particular reference to sections 3-11 to 3-28 and 10-1 to 10-17.

[0317] Positive selection of the transduced cells can also be performed using magnetic separation of cells based on expression or a particular cell surface marker. In such separation techniques, cells to be positively selected are first contacted with specific binding agent (e.g., an antibody or reagent the interacts specifically with the cell surface marker). The cells are then contacted with retrievable particles (e.g., magnetically responsive particles) which are coupled with a reagent that binds the specific binding agent (that has bound to the positive cells). The cell-binding agent-particle complex can then be physically separated from non-labeled cells, for example using a magnetic field. When using magnetically responsive particles, the labeled cells can be retained in a container using a magnetic filed while the negative cells are removed. These and similar separation procedures are known to those of ordinary skill in the art.

[0318] Expression of the vector in the selected transduced cells can be assessed by a number of assays known to those skilled in the art. For example, Western blot or Northern analysis can be employed depending on the nature of the inserted nucleotide sequence of interest. Once expression has been established and the transformed T cells have been tested for the presence of the selected synthetic expression cassette, they are ready for infusion into a patient via the peripheral blood stream.

[0319] The invention includes a kit for genetic modification of an ex vivo population of primary mammalian cells. The kit typically contains a gene transfer vector coding for at least one selectable marker and at least one synthetic expression cassette contained in one or more containers, ancillary reagents or hardware, and instructions for use of the kit.

[0320] 2.4.3 Further Delivery Regimes

[0321] Any of the polynucleotides (e.g., expression cassettes) or polypeptides described herein (delivered by any of the methods described above) can also be used in combination with other DNA delivery systems and/or protein delivery systems. Non-limiting examples include co-administration of these molecules, for example, in prime-boost methods where one or more molecules are delivered in a "priming" step and, subsequently, one or more molecules are delivered in a "boosting" step. In certain embodiments, the delivery of one or more nucleic acid-containing compositions and is followed by delivery of one or more nucleic acid-containing compositions and/or one or more polypeptide-containing compositions (e.g., polypeptides comprising HIV antigens). In other embodiments, multiple nucleic acid "primes" (of the same or different nucleic acid molecules) can be followed by multiple polypeptide "boosts" (of the same or different polypeptides). Other examples include multiple nucleic acid administrations and multiple polypeptide administrations.

[0322] In any method involving co-administration, the various compositions can be delivered in any order. Thus, in embodiments including delivery of multiple different compositions or molecules, the nucleic acids need not be all delivered before the polypeptides. For example, the priming step may include delivery of one or more polypeptides and the boosting comprises delivery of one or more nucleic acids and/or one more polypeptides. Multiple polypeptide administrations can be followed by multiple nucleic acid administrations or polypeptide and nucleic acid administrations can be performed in any order. In any of the embodiments described herein, the nucleic acid molecules can encode all, some or none of the polypeptides. Thus, one or more or the nucleic acid molecules (e.g., expression cassettes) described herein and/or one or more of the polypeptides described herein can be co-administered in any order and via any administration routes. Therefore, any combination of polynucleotides and/or polypeptides described herein can be used to generate elicit an immune reaction.

[0323] 3.0 Improved HIV-1 Gag and Pol Expression Cassettes

[0324] While not desiring to be bound by any particular model, theory, or hypothesis, the following information is presented to provide a more complete understanding of the present invention.

[0325] The world health organization (WHO) estimated the number of people worldwide that are infected with HIV-1 to exceed 36.1 million. The development of a safe and effective HIV vaccine is therefore essential at this time. Recent studies have demonstrated the importance of CTL in controlling the HIV-1 replication in infected patients. Furthermore, CTL reactivity with multiple HIV antigens will be necessary for the effective control of virus replication. Experiments performed in support of the present invention suggest that the inclusion of HIV-1 Gag and Pol, beside Env for the induction of neutralizing antibodies, into the vaccine is useful.

[0326] To increase the potency of HIV-1 vaccine candidates, codon modified Gag and Pol expression cassettes were designed, either for Gag alone or Gag plus Pol. To evaluate possible differences in expression and potency, the expression of these constructs was analyzed and immunogenicity studies carried out in mice.

[0327] Several expression cassettes encoding Gag and Pol were designed, including, but not limited to, the following: GagProtease, GagPol.DELTA.integrase with frameshift (gagFSpol), and GagPol.DELTA.integrase in-frame (gagpol). Versions of GagPol.DELTA.integrase in-frame were also designed with attenuated (Att) or non-functional Protease (Ina). The nucleic acid sequences were codon modified to correspond to the codon usage of highly expressed human genes. Mice were immunized with titrated DNA doses and humoral and cellular immune responses evaluated by ELISA and intracellular cytokine staining (Example 10).

[0328] The immune responses in mice has been seen to be correlated with relative levels of expression in vitro. Vaccine studies in rhesus monkeys will further address immune responses and expression levels in vivo.

[0329] 4.0 Enhanced Vaccine Technologies for the Induction of Potent Neutralizing Antibodies and Cellular Immune Responses Against HIV.

[0330] While not desiring to be bound by any particular model, theory, or hypothesis, the following information is presented to provide a more complete understanding of the present invention.

[0331] Protection against HIV infection will likely require potent and broadly reactive pre-existing neutralizing antibodies in vaccinated individuals exposed to a virus challenge. Although cellular immune responses are desirable to control viremia in those who get infected, protection against infection has not been demonstrated for vaccine approaches that rely exclusively on the induction of these responses. For this reason, experiments performed in support of the present invention use prime-boost approaches that employ novel V-deleted envelope antigens from primary HIV isolates (e.g., R5 subtype B (HIV-1.sub.SF162) and subtype C (HIV-1.sub.TVI) strains). These antigens were delivered by enhanced DNA [polyactide co-glycolide (PLG) microparticle formulations or electroporation] or alphavirus replicon particle-based vaccine approaches, followed by booster immunizations with Env proteins in MF59 adjuvant. Efficient in vivo expression of plasmid encoded genes by electrical permeabilization has been described (see, e.g., Zucchelli et al. (2000) J. Virol. 74:11598-11607; Banga et al. (1998) Trends Biotechnol. 10:408-412; Heller et al. (1996) Febs Lett. 389:225-228; Mathiesen et al. (1999) Gene Ther. 4:508-514; Mir et al. (1999) Proc. Nat'l Acad. Sci. USA 8:4262-4267; Nishi et al. (1996) Cancer Res. 5:1050-1055). Both native and V-deleted monomeric (gp120) and oligomeric (o-gp140) forms of protein from the SF162 strain were tested as boosters. All protein preparations were highly purified and extensively characterized by biophysical and immunochemical methodologies. Results from rabbit and primate immunogenicity studies indicated that, whereas neutralizing antibody responses could be consistently induced against the parental non-V2-deleted SF162 virus, the induction of responses against heterologous HIV strains improved with deletion of the V2 loop of the immunogens. Moreover, using these prime-boost vaccine regimens, potent HIV antigen-specific CD4+ and CD8+ T-cell responses were also demonstrated.

[0332] Based on these findings, V2-deleted envelope DNA and protein vaccines were chosen for advancement toward clinical evaluation. Similar approaches for immunization may be employed using, for example, nucleic acid immunization employing the synthetic HIV polynucleotides of the present invention coupled with corresponding or heterologous HIV-derived polypeptide boosts.

[0333] One embodiment of this aspect of the present invention may be described generally as follows. Antigens are selected for the vaccine composition(s). Env polypeptides are typically employed in a first antigenic composition used to induce an immune response. Further, Gag polypeptides are typically employed in a second antigenic composition used to induce an immune response. The second antigenic composition may include further HIV-derived polypeptide sequences, including, but not limited to, Pol, Tat, Rev, Nef, Vif, Vpr, and/or Vpu sequences. A DNA prime vaccination is typically performed with the first and second antigenic compositions. Further DNA vaccinations with one or more of the antigenic compositions may also be included at selected time intervals. The prime is typically followed by at least one boost. The boost may, for example, include adjuvanted HIV-derived polypeptides (e.g., corresponding to those used for the DNA vaccinations), coding sequences for HIV-derived polypeptides (e.g., corresponding to those used for the DNA vaccinations) encoded by a viral vector, further DNA vaccinations, and/or combinations of the foregoing. In one embodiment, a DNA prime is administered with a first antigenic composition (e.g., a DNA construct encoding an Envelope polypeptide) and second antigenic composition (e.g., a DNA construct encoding a Gag polypeptide, a Pol polypeptide, a Tat polypeptide, a Nef polypeptide, and a Rev polypeptide). The DNA construct for use in the prime may, for example, comprise a CMV promoter operably linked to the polynucleotide encoding the polypeptide sequence. The DNA prime is followed by a boost, for example, an adjuvanted Envelope polypeptide boost and a viral vector boost (where the viral vector encodes, e.g., a Gag polypeptide, a Pol polypeptide, a Tat polypeptide, a Nef polypeptide, and a Rev polypeptide). Alternately (or in addition), the boost may be an adjuvanted Gag polypeptide, Pol polypeptide, Tat polypeptide, Nef polypeptide, and Rev polypeptide boost and a viral vector boost (where the viral vector encodes, e.g., an Envelope polypeptide). The boost may include all polypeptide antigens which were encoded in the DNA prime; however, this is not required. Further, different polypeptide antigens may be used in the boost relative to the initial vaccination and visa versa. Further, the initial vaccination may be a viral vector rather than a DNA construct.

[0334] Some factors that may be considered in HIV envelope vaccine design are as follows. Envelope-based vaccines have demonstrated protection against infection in non-human primate models. Passive antibody studies have demonstrated protection against HIV infection in the presence of neutralizing antibodies against the virus challenge stock. Vaccines that exclude Env generally confer less protective efficacy. Experiments performed in support of the present invention have demonstrated that monomeric gp120 protein-derived from the SF2 lab strain provided neutralization of HIV-1 lab strains and protection against virus challenges in primate models. Primary gp120 protein derived from Thai E field strains provided cross-subtype neutralization of lab strains. Primary sub-type B oligomeric o-gp140 protein provided partial neutralization of subtype B primary (field) isolates. Primary sub-type B o-gp140.DELTA.V2 DNA prime plus protein boost provided potent neutralization of diverse subtype B primary isolates and protection against virus challenge in primate models. Primary sub-type C o-gp140 and o-gp140.DELTA.V2 likely provide similar results to those just described for sub-type B.

[0335] Vaccine strategies for induction of potent, broadly reactive, neutralizing antibodies may be assisted by construction of Envelope polypeptide structures that expose conserved neutralizing epitopes, for example, variable-region deletions and de-glycosylations, envelope protein-receptor complexes, rational design based on crystal structure (e.g., .beta.-sheet deletions), and gp41-fusion domain based immunogens.

[0336] Stable CHO cell lines for envelope protein production have been developed using optimized envelope polypeptide coding sequences, including, but not limited to, the following: gp120, o-gp140, gp120.DELTA.V2, o-gp140.DELTA.V2, gp120.DELTA.V1V2, o-gp140.DELTA.V1V2.

[0337] In addition, following prime-boost regimes (such as those described above) appear to be beneficial to help reduce viral load in infected subjects, as well as possibly slow or prevent progression of HIV-related disease (relative to untreated subjects).

[0338] Exemplary antigenic compositions and immunogenicity studies are presented in Examples 9, 10, 11, and 12.

EXPERIMENTAL

[0339] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0340] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

Example 1

Generation of Synthetic Expression Cassettes

A. Generating Synthetic Polynucleotides

[0341] The polynucleotide sequences of the present invention were manipulated to maximize expression of their gene products. The order of the following steps may vary.

[0342] First, the HIV-1 codon usage pattern was modified so that the resulting nucleic acid coding sequence was comparable to codon usage found in highly expressed human genes. The HIV codon usage reflects a high content of the nucleotides A or T of the codon-triplet. The effect of the HIV-1 codon usage is a high AT content in the DNA sequence that results in a high AU content in the RNA and in a decreased translation ability and instability of the mRNA. In comparison, highly expressed human codons prefer the nucleotides G or C. The wild-type sequences were modified to be comparable to codon usage found in highly expressed human genes.

[0343] Second, for some genes non-functional variants were created. In the following table (Table B) mutations affecting the activity of several HIV genes are disclosed. All references cited are herein incorporated by reference. TABLE-US-00002 TABLE B Gene "Region" Exemplary Mutations Pol prot Att = Reduced activity by attenuation of Protease (Thr26Ser) (e.g., Konvalinka et al., 1995, J Virol 69: 7180-86) Ina = Mutated Protease, nonfunctional enzyme (Asp25Ala)(e.g., Konvalinka et al., 1995, J Virol 69: 7180-86) RT YM = Deletion of catalytic center (YMDD_AP; SEQ ID NO: 7) (e.g., Biochemistry, 1995, 34, 5351, Patel et. al.) WM = Deletion of primer grip region (WMGY_PI; SEQ ID NO: 8)) (e.g., J Biol Chem, 272, 17, 11157, Palaniappan, et. al., 1997) RNase no direct mutations, RnaseH is affected by "WM" mutation in RT Integrase 1) Mutation of HHCC domain, Cys40Ala (e.g., Wiskerchen et. al., 1995, J Virol, 69: 376). 2.) Inactivation catalytic center, Asp64Ala, Asp116Ala, Glu152Ala (e.g., Wiskerchen et. al., 1995, J Virol, 69: 376). 3) Inactivation of minimal DNA binding domain (MDBD), deletion of Trp235(e.g., Ishikawa et. al., 1999, J Virol, 73: 4475). Constructs int.opt.mut.SF2 and int.opt.mut_C (South Africa TV1) both contain all these mutations (1, 2, and 3) Env Mutations in cleavage site (e.g., mut1-4, 7) Mutations in glycosylation site (e.g., GM mutants, for example, change Q residue in V1 and/or V2 to N residue; may also be designated by residue altered in sequence) Tat Mutants of Tat in transactivation domain (e.g., Caputo et al., 1996, Gene Ther. 3: 235) cys22 mutant (Cys22Gly) = TatC22 cys37 mutant (Cys37Ser) = TatC37 cys22/37 double mutant = TatC22/37 Rev Mutations in Rev domains (e.g., Thomas et al., 1998, J Virol. 72: 2935-44) Mutation in RNA binding-nuclear localization ArgArg38, 39AspLeu = M5 Mutation in activation domain LeuGlu78, 79AspLeu = M10 Nef Mutations of myristoyilation signal and in oligomerization domain: 1. Single point mutation myristoyilation signal: Gly-to-Ala = -Myr 2. Deletion of N-terminal first 18 (sub-type B, e.g., SF162) or 19 (sub-type C, e.g., South Africa clones) amino acids: -Myr18 or -Myr19 (respectively) (e.g., Peng and Robert-Guroff, 2001, Immunol Letters 78: 195-200) Single point mutation oligomerization: (e.g., Liu et al., 2000, J Virol 74: 5310-19) Asp125Gly (sub B SF162) or Asp124Gly (sub C South Africa clones) Mutations affecting (1) infectivity (replication) of HIV-virions and/or (2) CD4 down regulation. (e.g., Lundquist et al. (2002) J Virol. 76(9): 4625-33) Vif Mutations of Vif: e.g., Simon et al., 1999, J Virol 73: 2675-81 Vpr Mutations of Vpr: e.g., Singh et al., 2000, J Virol 74: 10650-57 Vpu Mutations of Vpu: e.g., Tiganos et al., 1998, Virology 251: 96-107

[0344] Constructs comprising some of these mutations are described herein. Vif, vpr and vpu synthetic constructs are described. Reducing or eliminating the function of the associated gene products can be accomplished employing the teachings set forth in the above table, in view of the teachings of the present specification.

[0345] In one embodiment of the invention, the full length coding region of the Gag-polymerase sequence is included with the synthetic Gag sequences in order to increase the number of epitopes for virus-like particles expressed by the synthetic, optimized Gag expression cassette. Because synthetic HIV-1 Gag-polymerase expresses the potentially deleterious functional enzymes reverse transcriptase (RT) and integrase (INT) (in addition to the structural proteins and protease), it is important to inactivate RT and INT functions. Several in-frame deletions in the RT and INT reading frame can be made to achieve catalytic nonfunctional enzymes with respect to their RT and INT activity. {Jay. A. Levy (Editor) (1995) The Retroviridae, Plenum Press, New York. ISBN 0-306-45033.times.. Pages 215-20; Grimison, B. and Laurence, J. (1995), Journal Of Acquired Immune Deficiency Syndromes and Human Retrovirology 9(1):58-68; Wakefield, J. K., et al., (1992) Journal Of Virology 66(11):6806-6812; Esnouf, R., et al., (1995) Nature Structural Biology 2(4):303-308; Maignan, S., et al., (1998) Journal Of Molecular Biology 282(2):359-368; Katz, R. A. and Skalka, A. M. (1994) Annual Review Of Biochemistry 73 (1994); Jacobo-Molina, A., et al., (1993) Proceedings Of the National Academy Of Sciences Of the United States Of America 90(13):6320-6324; Hickman, A. B., et al., (1994) Journal Of Biological Chemistry 269(46):29279-29287; Goldgur, Y., et al., (1998) Proceedings Of the National Academy Of Sciences Of the United States Of America 95(16):9150-9154; Goette, M., et al., (1998) Journal Of Biological Chemistry 273(17):10139-10146; Gorton, J. L., et al., (1998) Journal of Virology 72(6):5046-5055; Engelman, A., et al., (1997) Journal Of Virology 71(5):3507-3514; Dyda, F., et al., Science 266(5193):1981-1986; Davies, J. F., et al., (1991) Science 252(5002):88-95; Bujacz, G., et al., (1996) Febs Letters 398(2-3):175-178; Beard, W. A., et al., (1996) Journal Of Biological Chemistry 271(21):12213-12220; Kohlstaedt, L. A., et al., (1992) Science 256(5065):1783-1790; Krug, M. S. and Berger, S. L. (1991) Biochemistry 30(44):10614-10623; Mazumder, A., et al., (1996) Molecular Pharmacology 49(4):621-628; Palaniappan, C., et al., (1997) Journal Of Biological Chemistry 272(17): 11157-11164; Rodgers, D. W., et al., (1995) Proceedings Of the National Academy Of Sciences Of the United States Of America 92(4): 1222-1226; Sheng, N. and Dennis, D. (1993) Biochemistry 32(18):4938-4942; Spence, R. A., et al., (1995) Science 267(5200):988-993.}

[0346] Furthermore selected B- and/or T-cell epitopes can be added to the Gag-polymerase constructs within the deletions of the RT- and INT-coding sequence to replace and augment any epitopes deleted by the functional modifications of RT and INT. Alternately, selected B- and T-cell epitopes (including CTL epitopes) from RT and INT can be included in a minimal VLP formed by expression of the synthetic Gag or synthetic GagProt cassette, described above. (For descriptions of known HIV B- and T-cell epitopes see, HIV Molecular Immunology Database CTL Search Interface; Los Alamos Sequence Compendia, 1987-1997; Internet address: http://hiv-web.lanl.gov/immunology/index.html.)

[0347] In another aspect, the present invention comprises Env coding sequences that include, but are not limited to, polynucleotide sequences encoding the following HIV-encoded polypeptides: gp160, gp140, and gp120 (see, e.g., U.S. Pat. No. 5,792,459 for a description of the HIV-1.sub.SF2 ("SF2") Env polypeptide). The relationships between these polypeptides is shown schematically in FIG. 3 (in the figure: the polypeptides are indicated as lines, the amino and carboxy termini are indicated on the gp160 line; the open circle represents the oligomerization domain; the open square represents a transmembrane spanning domain (TM); and "c" represents the location of a cleavage site, in gp140.mut the "X" indicates that the cleavage site has been mutated such that it no longer functions as a cleavage site). The polypeptide gp160 includes the coding sequences for gp120 and gp41. The polypeptide gp41 is comprised of several domains including an oligomerization domain (OD) and a transmembrane spanning domain (TM). In the native envelope, the oligomerization domain is required for the non-covalent association of three gp41 polypeptides to form a trimeric structure: through non-covalent interactions with the gp41 trimer (and itself), the gp120 polypeptides are also organized in a trimeric structure. A cleavage site (or cleavage sites) exists approximately between the polypeptide sequences for gp120 and the polypeptide sequences corresponding to gp41. This cleavage site(s) can be mutated to prevent cleavage at the site. The resulting gp140 polypeptide corresponds to a truncated form of gp160 where the transmembrane spanning domain of gp41 has been deleted. This gp140 polypeptide can exist in both monomeric and oligomeric (i.e. trimeric) forms by virtue of the presence of the oligomerization domain in the gp41 moiety. In the situation where the cleavage site has been mutated to prevent cleavage and the transmembrane portion of gp41 has been deleted the resulting polypeptide product is designated "mutated" gp140 (e.g., gp140.mut). As will be apparent to those in the field, the cleavage site can be mutated in a variety of ways. (See, also, WO 00/39302).

[0348] Wild-type HIV coding sequences (e.g., Gag, Env, Pol, tat, rev, nef, vpr, vpu, vif, etc.) can be selected from any known HIV isolate and these sequences manipulated to maximize expression of their gene products following the teachings of the present invention. The wild-type coding region maybe modified in one or more of the following ways. In one embodiment, sequences encoding hypervariable regions of Env, particularly V1 and/or V2 were deleted. In other embodiments, mutations were introduced into sequences, for example, encoding the cleavage site in Env to abrogate the enzymatic cleavage of oligomeric gp140 into gp120 monomers. (See, e.g., Earl et al. (1990) PNAS USA 87:648-652; Earl et al. (1991) J. Virol. 65:31-41). In yet other embodiments, hypervariable region(s) were deleted, N-glycosylation sites were removed and/or cleavage sites mutated. As discussed above, different mutations may be introduced into the coding sequences of different genes (see, e.g., Table B). For example, Tat coding sequences were modified according to the teachings of the present specification, for example to affect the transactivation domain of the gene product (e.g., replacing a cystein residue at position 22 with a glycine, Caputo et al. (1996) Gene Therapy 3:235).

[0349] To create the synthetic coding sequences of the present invention the gene cassettes are designed to comprise the entire coding sequence of interest. Synthetic gene cassettes are constructed by oligonucleotide synthesis and PCR amplification to generate gene fragments. Primers are chosen to provide convenient restriction sites for subcloning. The resulting fragments are then ligated to create the entire desired sequence which is then cloned into an appropriate vector. The final synthetic sequences are (i) screened by restriction endonuclease digestion and analysis, (ii) subjected to DNA sequencing in order to confirm that the desired sequence has been obtained and (iii) the identity and integrity of the expressed protein confirmed by SDS-PAGE and Western blotting. The synthetic coding sequences are assembled at Chiron Corp. (Emeryville, Calif.) or by the Midland Certified Reagent Company (Midland, Tex.).

[0350] Percent identity to the synthetic sequences of the present invention can be determined, for example, using the Smith-Waterman search algorithm (Time Logic, Incline Village, Nev.), with the following exemplary parameters: weight matrix=nuc4.times.4hb; gap opening penalty=20, gap extension penalty=5, reporting threshold=1; alignment threshold=20.

[0351] Various forms of the different embodiments of the present invention (e.g., constructs) may be combined.

[0352] Exemplary embodiments of the synthetic polynucleotides of the present invention include, but are not limited to, the sequences presented in Table C. TABLE-US-00003 TABLE C Type B Synthetic, Codon Optimized Polynucleotides FIG. Num- Name ber Description (encoding) GagComplPolmut.SF2 6 Gag complete, RT mutated, (SEQ ID NO: 9) Protease functional; all in frame GagComplPolmutAtt.SF2 7 Gag complete, RT mutated, (SEQ ID NO: 10) Protease attenuated; all in frame GagComplPolmutIna.SF2 8 Gag complete, RT mutated, (SEQ ID NO: 11) Protease non-functional; all in frame gagCpolInaTatRevNef.opt_B 9 Gag complete, protease (SEQ ID NO: 12) non-functional, RT mutated, tat mutated, rev mutated, nef mutated; all in frame GagPolmutAtt.SF2 10 Gag, RT mutated, Protease (SEQ ID NO: 13) attenuated; all in frame GagPolmutIna.SF2 11 Gag, RT mutated, Protease (SEQ ID NO: 14) non-functional; all in frame GagProtInaRTmut.SF2 12 Gag, Protease (SEQ ID NO: 15) non-functional, RT mutated; all in frame GagProtInaRTmutTatRevNef.opt_B 13 Gag, protease (SEQ ID NO: 16) non-functional, RT mutated, tat mutated, rev mutated, nef mutated; all in frame GagRTmut.SF2 14 Gag, RT mutated; all in (SEQ ID NO: 17) frame GagTatRevNef.opt_B 15 Gag, tat mutated, rev (SEQ ID NO: 18) mutated, nef mutated; all in frame gp140.modSF162.CwtLmod 16 gp140 derived from SF162 (SEQ ID NO: 19) with a HIV Type C (TV1) optimized leader sequence gp140.modSF162.CwtLnat 17 gp140 derived from SF162 (SEQ ID NO: 20) with a HIV Type C (TV1) native leader sequence gp160.modSF162.delV2.mut7 18 gp160 derived from SF162, (SEQ ID NO: 21) deletion of V2 loop, mutated cleavage site gp160.modSF162.delV2.mut8 19 gp160 derived from SF162, (SEQ ID NO: 22) deletion of V2 loop, mutated cleavage site int.opt.mut.SF2 20 integrase mutated (SEQ ID NO: 23) int.opt.SF2 21 integrase (SEQ ID NO: 24) nef.D125G.-myr.opt.SF162 22 nef mutated, (SEQ ID NO: 25) myristoyilation defective nef.D107G.-myr18.opt.SF162 23 nef mutated, (SEQ ID NO: 26) myristoyilation defective nef.opt.D125G.SF162 24 nef mutated (SEQ ID NO: 27) nef.opt.SF162 25 nef (SEQ ID NO: 28) p15RnaseH.opt.SF2 26 p15 RNase H; in-frame (SEQ ID NO: 29) p2Pol.opt.YMWM.SF2 27 p2 pol mutated (SEQ ID NO: 30) (RT YM, WM) p2PolInaopt.YM.SF2 28 p2 pol, protease non- (SEQ ID NO: 31) functional, RT YM; all in frame p2Polopt.SF2 29 p2 pol; all in frame (SEQ ID NO: 32) p2PolTatRevNef.opt.native_B 30 p2 pol tat rev nef; all (SEQ ID NO: 33) native; all in frame p2PolTatRevNef.opt_B 31 p2 pol, protease mutated, (SEQ ID NO: 34) RT mutated, tat mutated, rev mutated, nef, mutated; all in frame pol.opt.SF2 32 pol (SEQ ID NO: 35) prot.opt.SF2 33 protease (SEQ ID NO: 36) protIna.opt.SF2 34 protease non-functional (SEQ ID NO: 37) protInaRT.YM.opt.SF2 35 protease non-functional, RT (SEQ ID NO: 38) YM mutated; all in frame protInaRT.YMWM.opt.SF2 36 protease non-functional, RT (SEQ ID NO: 39) YM WM mutated; all in frame ProtInaRTmut.SF2 37 Protease inactive, RT (SEQ ID NO: 40) mutated; all in frame protRT.opt.SF2 38 protease RT; all in frame (SEQ ID NO: 41) ProtRT.TatRevNef.opt_B 39 protease mutated, RT (SEQ ID NO: 42) mutated, tat mutated, rev mutated, nef, mutated; all in frame ProtRTTatRevNef.opt_B 40 protease mutated, RT (SEQ ID NO: 43) mutated, tat mutated, rev mutated, nef, mutated; all in frame rev.exon1_2.M5-10.opt.SF162 41 rev exon 1 and 2 in-frame, (SEQ ID NO: 44) rev mutated rev.exon1_2.opt.SF162 42 rev exon 1 and 2 in-frame (SEQ ID NO: 45) RT.opt.SF2 (mutant) 43 RT mutant (SEQ ID NO: 46) RT.opt.SF2 (native) 44 RT native (SEQ ID NO: 47) RTmut.SF2 45 RT mutated (SEQ ID NO: 48) tat.exon1_2.opt.C22-37.SF2 46 tat exon 1 and 2 in-frame, (SEQ ID NO: 49) tat mutated tat.exon1_2.opt.C37.SF2 47 tat exon 1 and 2 in-frame, (SEQ ID NO: 50) tat mutated TatRevNef.opt.native.SF162 48 tat native, rev native, (SEQ ID NO: 51) nef native; all in frame TatRevNef.opt.SF162 49 tat mutated, rev mutated, (SEQ ID NO: 52) nef mutated; all in frame TatRevNefGag B 50 tat mutated, rev mutated, (SEQ ID NO: 53) nef mutated, gag; all in frame TatRevNefgagCpolIna B 51 tat mutated, rev mutated, (SEQ ID NO: 54) nef mutated, gag complete, protease non-functional, RT mutated; all in frame TatRevNefGagProtInaRTmut B 52 tat mutated, rev mutated, (SEQ ID NO: 55) nef mutated, gag, protease non-functional, RT mutant; all in frame TatRevNefp2Pol.opt_B 53 tat mutated, rev mutated, (SEQ ID NO: 56) nef mutated, p2 pol, protease mutated, RT mutated; all in frame TatRevNefprotRTopt B 54 tat mutated, rev mutated, (SEQ ID NO: 57) nef mutated, protease mutated, RT mutated; all in frame vif.opt.SF2 55 optimized vif derived (SEQ ID NO: 58) from SF2 vpr.opt.SF2 56 optimized vpr derived from (SEQ ID NO: 59) SF2 vpu.opt.SF162 57 optimized vpu derived from (SEQ ID NO: 60) SF162 {In Table C, .mut or .mut7 or .mut8 = envelope mutated in cellular protease cleavage site between gp120/gp41 (i.e., to prevent cleavage; e.g., better for purifying protein)}

B. Creating Expression Cassettes Comprising the Synthetic Polynucleotides of the Present Invention.

[0353] The synthetic DNA fragments of the present invention are cloned into the following expression vectors: pCMVKm2, for transient expression assays and DNA immunization studies, the pCMVKm2 vector was derived from pCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) and comprises a kanamycin selectable marker, a ColE1 origin of replication, a CMV promoter enhancer and Intron A, followed by an insertion site for the synthetic sequences described below followed by a polyadenylation signal derived from bovine growth hormone--the pCMVKm2 vector differs from the pCMV-link vector only in that a polylinker site was inserted into pCMVKm2 to generate pCMV-link; pESN2dhfr and pCMVPLEdhfr (also known as pCMVIII), for expression in Chinese Hamster Ovary (CHO) cells; and, pAcC13, a shuttle vector for use in the Baculovirus expression system (pAcC13, was derived from pAcC12 which was described by Munemitsu S., et al., Mol Cell Biol. 10(11):5977-5982, 1990). See, also co-owned WO 00/39302, WO 00/39303, WO 00/39304, and WO 02/04493, for a description of these vectors, all herein incorporated by reference in their entireties.

[0354] Briefly, construction of pCMVPLEdhfr (pCMVIII) was as follows. To construct a DHFR cassette, the EMCV IRES (internal ribosome entry site) leader was PCR-amplified from pCite-4a+ (Novagen, Inc., Milwaukee, Wis.) and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as an Xba-Nco fragment to give pET-EMCV. The dhfr gene was PCR-amplified from pESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place of the translation stop codon and inserted as an Nco-BamH1 fragment to give pET-E-DHFR. Next, the attenuated neo gene was PCR amplified from a pSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into the unique BamH1 site of pET-E-DHFR to give pET-E-DHFR/Neo.sub.(m2). Then, the bovine growth hormone terminator from pCDNA3 (Invitrogen, Inc., Carlsbad, Calif.) was inserted downstream of the neo gene to give pET-E-DHFR/Neo.sub.(m2)BGHt. The EMCV-dhfr/neo selectable marker cassette fragment was prepared by cleavage of pET-E-DHFR/Neo.sub.(m2)BGHt. The CMV enhancer/promoter plus Intron A was transferred from pCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-Sal1 fragment into pUC19 (New England Biolabs, Inc., Beverly, Mass.). The vector backbone of pUC19 was deleted from the Nde1 to the Sap1 sites. The above described DHFR cassette was added to the construct such that the EMCV IRES followed the CMV promoter to produce the final construct. The vector also contained an amp.sup.r gene and an SV40 origin of replication.

[0355] Expression vectors of the present invention contain one or more of the synthetic coding sequences disclosed herein, e.g., shown in the Figures. When the expression cassette contains more than one coding sequence the coding sequences may all be in-frame to generate one polyprotein; alternately, the more than one polypeptide coding sequences may comprise a polycistronic message where, for example, an IRES is placed 5' to each polypeptide coding sequence.

Example 2

Expression Assays for the Synthetic Coding Sequences

[0356] The wild-type sequences are cloned into expression vectors having the same features as the vectors into which the synthetic HIV-derived sequences were cloned.

[0357] Expression efficiencies for various vectors carrying the wild-type (any known isolated) and corresponding synthetic sequence(s) are evaluated as follows. Cells from several mammalian cell lines (293, RD, COS-7, and CHO; all obtained from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209) are transfected with 2 .mu.g of DNA in transfection reagent LT1 (PanVera Corporation, 545 Science Dr., Madison, Wis.). The cells are incubated for 5 hours in reduced serum medium (Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The medium is then replaced with normal medium as follows: 293 cells, IMDM, 10% fetal calf serum, 2% glutamine (BioWhittaker, Walkersville, Md.); RD and COS-7 cells, D-MEM, 10% fetal calf serum, 2% glutamine (Opti-MEM, Gibco-BRL, Gaithersburg, Md.); and CHO cells, Ham's F-12, 10% fetal calf serum, 2% glutamine (Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The cells are incubated for either 48 or 60 hours. Supernatants are harvested and filtered through 0.45 .mu.m syringe filters and, optionally, stored at -20.degree. C.

[0358] Supernatants are evaluated using the Coulter p24-assay (Coulter Corporation, Hialeah, Fla., US), using 96-well plates coated with a suitable monoclonal antibody directed against an HIV antigen (e.g, a murine monoclonal directed again an HIV core antigen). The appropriate HIV antigen binds to the coated wells and biotinylated antibodies against HIV recognize the bound antigen. Conjugated strepavidin-horseradish peroxidase reacts with the biotin. Color develops from the reaction of peroxidase with TMB substrate. The reaction is terminated by addition of 4N H.sub.2SO.sub.4. The intensity of the color is directly proportional to the amount of HIV antigen in a sample.

[0359] Chinese hamster ovary (CHO) cells are also transfected with plasmid DNA encoding the synthetic HIV polypeptides described herein (e.g., pESN2dhfr or pCMVIII vector backbone) using Mirus TransIT-LT1 polyamine transfection reagent (Pan Vera) according to the manufacturers instructions and incubated for 96 hours. After 96 hours, media is changed to selective media (F12 special with 250 .mu.g/ml G418) and cells are split 1:5 and incubated for an additional 48 hours. Media is changed every 5-7 days until colonies start forming at which time the colonies are picked, plated into 96 well plates and screened by Capture ELISA. Positive clones are expanded in 24 well plates and are screened several times for HIV protein production by Capture ELISA, as described above. After reaching confluency in 24 well plates, positive clones are expanded to T25 flasks (Corning, Corning, N.Y.). These are screened several times after confluency and positive clones are expanded to T75 flasks.

[0360] Positive T75 clones are frozen in LN2 and the highest expressing clones are amplified with 0-5 .mu.M methotrexate (MTX) at several concentrations and plated in 100 mm culture dishes. Plates are screened for colony formation and all positive closed are again expanded as described above. Clones are expanded an amplified and screened at each step capture ELISA. Positive clones are frozen at each methotrexate level. Highest producing clones are grown in perfusion bioreactors (3 L, 100 L) for expansion and adaptation to low serum suspension culture conditions for scale-up to larger bioreactors.

[0361] Data from experiments performed in support of the present invention show that the synthetic HIV expression cassettes provided dramatic increases in production of their protein products, relative to the native (wild-type) sequences, when expressed in a variety of cell lines and that stably transfected CHO cell lines, which express the desired HIV polypeptide(s), may be produced. Production of HIV polypeptides using CHO cells provides (i) correct glycosylation patterns and protein conformation (as determined by binding to panel of MAbs); (ii) correct binding to CD4 receptor molecules; (iii) absence of non-mammalian cell contaminants (e.g., insect viruses and/or cells); and (iv) ease of purification.

Example 3

Western Blot Analysis of Expression

[0362] Western blot analysis of cells transfected with the HIV expression cassettes described herein are performed essentially as described in co-owned WO 00/39302. Briefly, human 293 cells are transfected as described in Example 2 with pCMV6a-based vectors containing native or synthetic HIV expression cassettes. Cells are cultivated for 60 hours post-transfection. Supernatants are prepared as described. Cell lysates are prepared as follows. The cells are washed once with phosphate-buffered saline, lysed with detergent [1% NP40 (Sigma Chemical Co., St. Louis, Mo.) in 0.1 M Tris-HCl, pH 7.5], and the lysate transferred into fresh tubes. SDS-polyacrylamide gels (pre-cast 8-16%; Novex, San Diego, Calif.) are loaded with 20 .mu.l of supernatant or 12.5 .mu.l of cell lysate. A protein standard is also loaded (5 .mu.l, broad size range standard; BioRad Laboratories, Hercules, Calif.). Electrophoresis is carried out and the proteins are transferred using a BioRad Transfer Chamber (BioRad Laboratories, Hercules, Calif.) to Immobilon P membranes (Millipore Corp., Bedford, Mass.) using the transfer buffer recommended by the manufacturer (Millipore), where the transfer is performed at 100 volts for 90 minutes. The membranes are exposed to HIV-1-positive human patient serum and immunostained using o-phenylenediamine dihydrochloride (OPD; Sigma).

[0363] The results of the immunoblotting analysis are used to show that cells containing the synthetic HIV expression cassette produce the expected HIV-polypeptide(s) at higher per-cell concentrations than cells containing the native expression cassette.

Example 4

In Vivo Immunogenicity of Synthetic HIV Expression Cassettes

A. Immunization

[0364] To evaluate the immunogenicity of the synthetic HIV expression cassettes, a mouse study may be performed. The plasmid DNA, e.g., pCMVKM2 carrying an expression cassette comprising a synthetic sequence of the present invention, is diluted to the following final concentrations in a total injection volume of 100 .mu.l: 20 .mu.g, 2 .mu.g, 0.2 .mu.g, and 0.02 .mu.g. To overcome possible negative dilution effects of the diluted DNA, the total DNA concentration in each sample is brought up to 20 .mu.g using the vector (pCMVKM2) alone. As a control, plasmid DNA comprising an expression cassette encoding the native, corresponding polypeptide is handled in the same manner. Twelve groups of four Balb/c mice (Charles River, Boston, Mass.) are intramuscularly immunized (50 .mu.l per leg, intramuscular injection into the tibialis anterior) using varying dosages.

B. Humoral Immune Response

[0365] The humoral immune response is checked with a suitable anti-HIV antibody ELISAs (enzyme-linked immunosorbent assays) of the mice sera 0 and 4 weeks post immunization (groups 5-12) and, in addition, 6 and 8 weeks post immunization, respectively, 2 and 4 weeks post second immunization (groups 1-4).

[0366] The antibody titers of the sera are determined by anti-HIV antibody ELISA. Briefly, sera from immunized mice were screened for antibodies directed against an appropriate HIV protein (e.g., HIV p55 for Gag). ELISA microtiter plates are coated with 0.2 .mu.g of HIV protein per well overnight and washed four times; subsequently, blocking is done with PBS-0.2% Tween (Sigma) for 2 hours. After removal of the blocking solution, 100 .mu.l of diluted mouse serum is added. Sera are tested at 1/25 dilutions and by serial 3-fold dilutions, thereafter. Microtiter plates are washed four times and incubated with a secondary, peroxidase-coupled anti-mouse IgG antibody (Pierce, Rockford, Ill.). ELISA plates are washed and 100 .mu.l of 3,3', 5,5'-tetramethyl benzidine (TMB; Pierce) was added per well. The optical density of each well is measured after 15 minutes. The titers reported are the reciprocal of the dilution of serum that gave a half-maximum optical density (O.D.).

[0367] The results of the mouse immunizations with plasmid-DNAs are used to show that the synthetic expression cassettes provide improvement of immunogenicity relative to the native expression cassettes. Also, the second boost immunization induces a secondary immune response after two weeks (groups 1-3).

C. Cellular Immune Response

[0368] The frequency of specific cytotoxic T-lymphocytes (CTL) is evaluated by a standard chromium release assay of peptide pulsed Balb/c mouse CD4 cells. HIV protein-expressing vaccinia virus infected CD-8 cells are used as a positive control (vv-protein). Briefly, spleen cells (Effector cells, E) are obtained from the BALB/c mice (immunized as described above). The cells are cultured, restimulated, and assayed for CTL activity against, e.g., Gag peptide-pulsed target cells as described (Doe, B., and Walker, C. M., AIDS 10(7):793-794, 1996). Cytotoxic activity is measured in a standard .sup.51Cr release assay. Target (T) cells are cultured with effector (E) cells at various E:T ratios for 4 hours and the average cpm from duplicate wells is used to calculate percent specific .sup.51Cr release.

[0369] Cytotoxic T-cell (CTL) activity is measured in splenocytes recovered from the mice immunized with HIV DNA constructs described herein. Effector cells from the DNA-immunized animals exhibit specific lysis of HIV peptide-pulsed SV-BALB (MHC matched) targets cells indicative of a CTL response. Target cells that are peptide-pulsed and derived from an MHC-unmatched mouse strain (MC57) are not lysed. The results of the CTL assays are used to show increased potency of synthetic HIV expression cassettes for induction of cytotoxic T-lymphocyte (CTL) responses by DNA immunization.

Example 5

In Vivo Immunogenicity of Synthetic HIV Expression Cassettes

A. General Immunization Methods

[0370] To evaluate the immunogenicity of the synthetic HIV expression cassettes, studies using guinea pigs, rabbits, mice, rhesus macaques and baboons are performed. The studies are typically structured as follows: DNA immunization alone (single or multiple); DNA immunization followed by protein immunization (boost); DNA immunization followed by Sindbis particle immunization; immunization by Sindbis particles alone.

B. Guinea Pigs

[0371] Experiments may be performed using guinea pigs as follows. Groups comprising six guinea pigs each are immunized intramuscularly or mucosally at 0, 4, and 12 weeks with plasmid DNAs encoding expression cassettes comprising one or more the sequences described herein. The animals are subsequently boosted at approximately 18 weeks with a single dose (intramuscular, intradermally or mucosally) of the HIV protein encoded by the sequence(s) of the plasmid boost and/or other HIV proteins. Antibody titers (geometric mean titers) are measured at two weeks following the third DNA immunization and at two weeks after the protein boost. These results are used to demonstrate the usefulness of the synthetic constructs to generate immune responses, as well as, the advantage of providing a protein boost to enhance the immune response following DNA immunization.

C. Rabbits

[0372] Experiments may be performed using rabbits as follows. Rabbits are immunized intramuscularly, mucosally, or intradermally (using a Bioject needless syringe) with plasmid DNAs encoding the HIV proteins described herein. The nucleic acid immunizations are followed by protein boosting after the initial immunization. Typically, constructs comprising the synthetic HIV-polypeptide-encoding polynucleotides of the present invention are highly immunogenic and generate substantial antigen binding antibody responses after only 2 immunizations in rabbits.

D. Humoral Immune Response

[0373] In any immunized animal model, the humoral immune response is checked in serum specimens from the immunized animals with an anti-HIV antibody ELISAs (enzyme-linked immunosorbent assays) at various times post-immunization. The antibody titers of the sera are determined by anti-HIV antibody ELISA as described above. Briefly, sera from immunized animals are screened for antibodies directed against the HIV polypeptide/protein(s) encoded by the DNA and/or polypeptide used to immunize the animals. Wells of ELISA microtiter plates are coated overnight with the selected HIV polypeptide/protein and washed four times; subsequently, blocking is done with PBS-0.2% Tween (Sigma) for 2 hours. After removal of the blocking solution, 100 .mu.l of diluted mouse serum is added. Sera are tested at 1/25 dilutions and by serial 3-fold dilutions, thereafter. Microtiter plates are washed four times and incubated with a secondary, peroxidase-coupled anti-mouse IgG antibody (Pierce, Rockford, Ill.). ELISA plates are washed and 100 .mu.l of 3,3',5,5'-tetramethyl benzidine (TMB; Pierce) was added per well. The optical density of each well is measured after 15 minutes. Titers are typically reported as the reciprocal of the dilution of serum that gave a half-maximum optical density (O.D.).

[0374] Cellular immune response may also be evaluated.

Example 6

DNA-Immunization of Baboons and Rhesus Macaques Using Expression Cassettes Comprising the Synthetic HIV Polynucleotides of the Present Invention

[0375] A. Baboons

[0376] Four baboons are immunized 3 times (weeks 0, 4 and 8) bilaterally, intramuscular into the quadriceps or mucosally using the gene delivery vehicles described herein. The animals are bled two weeks after each immunization and an HIV antibody ELISA is performed with isolated plasma. The ELISA is performed essentially as described above except the second antibody-conjugate is an anti-human IgG, g-chain specific, peroxidase conjugate (Sigma Chemical Co., St. Louis, Md. 63178) used at a dilution of 1:500. Fifty .mu.g/ml yeast extract may be added to the dilutions of plasma samples and antibody conjugate to reduce non-specific background due to preexisting yeast antibodies in the baboons. Lymphoproliferative responses to are observed in baboons two weeks post-fourth immunization (at week 14), and enhanced substantially post-boosting with HIV-polypeptide (at week 44 and 76). Such proliferation results are indicative of induction of T-helper cell functions.

[0377] B. Rhesus Macaques

[0378] The improved potency of the synthetic, codon-modified HIV-polypeptide encoding polynucleotides of the present invention, when constructed into expression plasmids may be confirmed in rhesus macaques. Typically, the macaques have detectable HIV-specific CTL after two or three 1 mg doses of modified HIV polynucleotide. In sum, these results demonstrate that the synthetic HIV DNA is immunogenic in non-human primates. Neutralizing antibodies may also detected.

Example 7

Co-Transfection of Monocistronic and Multicistronic Constructs

[0379] The present invention includes co-transfection with multiple, monocistronic expression cassettes, as well as, co-transfection with one or more multi-cistronic expression cassettes, or combinations thereof.

[0380] Such constructs, in a variety of combinations, may be transfected into 293T cells for transient transfection studies.

[0381] For example, a bicistronic construct may be made where the coding sequences for the different HIV polypeptides are under the control of a single CMV promoter and, between the two coding sequences, an IRES (internal ribosome entry site (EMCV IRES); Kozak, M., Critical Reviews in Biochemistry and Molecular Biology 27(45):385-402, 1992; Witherell, G. W., et al., Virology 214:660-663, 1995) sequence is introduced after the first HIV coding sequence and before the second HIV coding sequence.

[0382] Supernatants collected from cell culture are tested for the presence of the HIV proteins and indicate that appropriate proteins are expressed in the transfected cells (e.g., if an Env coding sequence was present the corresponding Env protein was detected; if a Gag coding sequence was present the corresponding Gag protein was detected, etc).

[0383] The production of chimeric VLPs by these cell lines may be determined using electron microscopic analysis. (See, e.g., co-owned WO 00/39302).

Example 8

Accessory Gene Components for an HIV-1 Vaccine: Functional Analysis of Mutated Tat, Rev and Nef Type C Antigens

[0384] The HIV-1 regulatory and accessory genes have received increased attention as components of HIV vaccines due to their role in viral pathogenesis, the high ratio of highly conserved CTL epitopes and their early expression in the viral life cycle. Because of various undesirable properties of these genes, questions regarding their safety and suitability as vaccine components have been raised. Experiments performed in support of the present invention have analyzed candidate HIV-1 subtype C tat, rev, and nef mutants for efficient expression and inactivation of potential deleterious functions. Other HIV subtype accessory genes may be evaluated similarly.

[0385] Sequence-modified, mutant tat, rev, and nef genes coding for consensus Tat, Rev and Nef proteins of South African HIV-1 subtype C were constructed using overlapping synthetic oligonucleotides and PCR-based site-directed mutagenesis. Constructs of the wild-type genes of the isolates closely resembling the respective consensus sequences were also made by PCR. In vitro expression of the constructs was analyzed by western blotting. The trans-activation activity of the Tat mutants and nuclear RNA export activity of the Rev mutants were studied after transfection of various cell lines using reporter-gene-based functionality assays.

[0386] In vitro expression of all constructs was demonstrated by western blotting using antigen specific mouse serum generated by DNA vaccination of mice with Tat, Rev, or Nef-expression plasmids. Expression levels of the sequence-modified genes were significantly higher than the wild-type genes.

[0387] Subtype B and C Tat cDNA was mutated to get TatC22, TatC37, and TatC22/37. Tat activity assays in three cell lines (RD, HeLa and 293). In the background of the subtype C consensus Tat, a single mutation at C22 was insufficient to inactivate LTR-dependent CAT expression. In contrast, this activity was significantly impaired in RD, 293 and HeLa cells using the single mutation, C37, or the double mutation, C22C37 (see Table B). Corresponding results were obtained for Tat mutants derived from subtype B strains.

[0388] Exemplary results are presented in FIG. 4 for transactivation activity of Tat mutants on LTR-CAT plasmid in 293 cells. Three independent assays were performed for each construct (FIG. 4, legend (1), (2), (3)).

[0389] The subtype C constructs TatC22ProtRTTatRevNef and ProtRTTatC22RevNef showed reduced Tat activity when compared to TatC22 alone, probably due to structural changes caused by the fusion protein.

[0390] For Rev constructs, to test for the loss of function, a CAT assay with a reporter plasmid including native or mutated Rev was used. As shown in FIG. 5, compared to wild-type Rev, the mRNA export function of the subtype C Rev with a double mutation, M5M10 (see Table B), was significantly lower. The background levels are shown in the "mock" data and the pDM128 reporter plasmid without Rev data. Two independent assays were performed for each construct (FIG. 5, legend (1), (2)).

[0391] Assays to measure Nef-specific functions may also be performed (Nef mutations are described in Table B). For example, FACs analysis is used to look for the presence of MHC1 and CD4 on cell surfaces. Cells are assayed in the presence and absence of Nef expression (for controls), as well as using the synthetic polynucleotides of the present invention that encode native nef protein and mutated nef protein. Down-regulation of MHC1 and CD4 expression indicates that the nef gene product is not functional, i.e., if nef is non-functional there is no down regulation.

[0392] These data demonstrate the impaired functionality of tat and rev DNA immunogens that may form part of a multi-component HIV-1 subtype C vaccine. In contrast to previous published data by other groups, the C22 mutation did not sufficiently inactivate the transactivation function of Tat. The C37 mutation appeared to be required for inactivation of subtype C and subtype B Tat proteins.

Example 9

Evaluation of Immunogenicity of Various HIV Polypeptide Encoding Plasmids

[0393] As noted above, the immunogenicity of any of the polynucleotides or expression cassettes described herein is readily evaluated. In the following table (Table D) are exemplified procedures involving a comparison of the immunogenicity of subtype B and C envelope plasmids, both individually and as a mixed-subtype vaccine, using electroporation, in rabbits. It will be apparent that such methods are equally applicable to any other HIV polypeptide. TABLE-US-00004 TABLE D Total Vol/ Sites/ Grp Animal Imm'n # Adjuvant Immunogen Dose Site Animal Route 1 1-4 1, 2 -- pCMV 160 TV1 DNA 1.0 mg 0.5 ml 2 IM/Quad (Electro) 3 -- pCMV 160 TV1 DNA 1.0 mg 0.5 ml 2 IM/Quad (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 2 5-8 1, 2 -- pCMV 160 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 -- pCMV 160 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 3 9-12 1, 2 -- pCMV 160 dV1/V2 1.0 mg 0.5 ml 2 IM/Quad TV1 DNA (Electro) 3 -- pCMV 160 dV1/V2 1.0 mg 0.5 ml 2 IM/Quad TV1 DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 4 13-16 1, 2 -- pCMV 140 TV1 DNA 1.0 mg 0.5 ml 2 IM/Quad (Electro) 3 -- pCMV 140 TV1 DNA 1.0 mg 0.5 ml 2 IM/Quad (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 5 17-20 1, 2 -- pCMV140dV2TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 -- pCMV140dV2TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 6 21-24 1, 2 -- pCMV 140 dV1/V2 1.0 mg 0.5 ml 2 IM/Quad TV1 DNA (Electro) 3 -- pCMV 140 dV1/V2 1.0 mg 0.5 ml 2 IM/Quad TV1 DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 7 25-28 1, 2 -- pSIN140dV2SF162 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 -- pSIN 140 dV2 SF162 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 8 29-32 1, 2 -- pCMV 140 dV2 1.0 mg 0.5 ml 2 IM/Quad SF162 DNA (Electro) 3 -- pCMV 140 dV2 1.0 mg 0.5 ml 2 IM/Quad SF162 DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 9 33-36 1, 2 -- pCMV 140 Q154 1.0 mg 0.5 ml 2 IM/Quad SF162 DNA (Electro) 3 -- pCMV 140 Q154 1.0 mg 0.5 ml 2 IM/Quad SF162 DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 10 37-40 1, 2 -- pCMV 140 dV2 1.0 mg SF162 DNA pCMV 140 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 -- pCMV 140 dV2 1.0 mg SF162 DNA pCMV 140 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 11 41-44 1, 2 -- pCMV 140 dV2 1.0 mg SF162 DNA pCMV 140 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 -- pCMV 140 dV2 1.0 mg SF162 DNA pCMV 140 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut

[0394] The MF59C adjuvant is a microfluidized emulsion containing 5% squalene, 0.5% tween 80, 0.5% span 85, in 10 mM citrate pH 6, stored in 10 mL aliquots at 4.degree. C.

[0395] Immunogens are prepared as described in the following table (Table E) for administration to animals in the various groups. Concentrations may vary from those described in the table, for example depending on the sequences and/or proteins being used. TABLE-US-00005 TABLE E Group Preparation 1-9 Immunization 1-3: pCMV and pSIN based plasmid DNA in Saline + Electroporation Subtype B and C plasmids will be provided frozen at a concentration of 1.0 mg/ml in sterile 0.9% saline. Store at -80.degree. C. until use. Thaw DNA at room temperature; the material should be clear or slightly opaque, with no particulate matter. Animals will be shaved prior to immunization, under sedation of 1x dose IP (by animal weight) of Ketamine - Xylazine (80 mg/ml - 4 mg/ml). Immunize each rabbit with 0.5 ml DNA mixture per side (IM/Quadriceps), 1.0 ml per animal. Follow the DNA injection with Electroporation using a 6-needle circular array with 1 cm diameter, 1 cm needle length. Electroporation pulses were given at 20 V/mm, 50 ms pulse length, 1 pulse/s. Immunization 3: Protein Immunization Proteins will be provided at 0.1 mg/ml in citrate buffer. Store at -80.degree. C. until use. Thaw at room temperature; material should be clear with no particulate matter. Add equal volume of MF59C adjuvant to thawed protein and mix well by inverting the tube. Immunize each rabbit with 0.5 ml adjuvanted protein per side, IM/Glut for a total of 1.0 ml per animal. Use material within 1 hour of the addition of adjuvant. Immunization 1-3: Combined subtype B and C plasmid DNA in Saline The immunogen will be provided at 2.0 mg/ml total DNA (1 mg/ml of each plasmid) in sterile 0.9% saline. Store at -80.degree. C. until use. Thaw DNA at room temperature; the material should be clear or slightly opaque, with no particulate matter. Animals will be shaved prior to immunization, under sedation of 1x dose IP (by animal weight) of Ketamine - Xylazine (80 mg/ml - 4 mg/ml). Immunize each rabbit with 0.5 ml DNA mixture per side (IM/Quadriceps), 1.0 ml per animal. Follow the DNA injection with Electroporation using a 6-needle circular array with 1 cm diameter, 1 cm needle length. Electroporation pulses were given at 20 V/mm, 50 ms pulse length, 1 pulse/s. 10-11 Immunization 3: Protein Immunization Proteins will be provided at 0.1 mg/ml in citrate buffer. Store at -80.degree. C. until use. Thaw at room temperature; material should be clear with no particulate matter. Add equal volume of MF59C adjuvant to thawed protein and mix well by inverting the tube. Immunize each rabbit with 0.5 ml adjuvanted protein per side, IM/Glut for a total of 1.0 ml per animal. Use material within 1 hour of the addition of adjuvant.

[0396] The immunization (Table F) and bleeding (Table G) schedules are as follows: TABLE-US-00006 TABLE F Imm'n: 1 2 3 3 Weeks: Group 0 4 16 16 1 pCMV 160 TV1 DNA pCMV 160 TV1 DNA pCMV 160 TV1 DNA Protein + MF59C 2 pCMV 160 dV2 TV1 DNA pCMV 160 dV2 TV1 DNA pCMV 160 dV2 TV1 DNA Protein + MF59C 3 pCMV 160 dV1/V2 TV1 DNA pCMV 160 dV1/V2 TV1 DNA pCMV 160 dV1/V2 TV1 DNA Protein + MF59C 4 pCMV 140 TV1 DNA pCMV 140 TV1 DNA pCMV 140 TV1 DNA Protein + MF59C 5 pCMV 140 dV2 TV1 DNA pCMV 140 dV2 TV1 DNA pCMV 140 dV2 TV1 DNA Protein + MF59C 6 pCMV 140 dV1/V2 TV1 DNA pCMV 140 dV1/V2 TV1 DNA pCMV 140 dV1/V2 TV1 DNA Protein + MF59C 7 pSIN 140 dV2 SF162 DNA pSIN 140 dV2 SF162 DNA pSIN 140 dV2 SF162 DNA Protein + MF59C 8 pCMV 140 dV2 SF162 DNA pCMV 140 dV2 SF162 DNA pCMV 140 dV2 SF162 DNA Protein + MF59C 9 pCMV 140 Q154 SF162 DNA pCMV 140 Q154 SF162 DNA pCMV 140 Q154 SF162 DNA Protein + MF59C 10 pCMV 140 dV2 SF162 DNA + pCMV pCMV 140 dV2 SF162 DNA + pCMV pCMV 140 dV2 SF162 DNA + pCMV Protein + MF59C 140 dV2 TV1 DNA 140 dV2 TV1 DNA 140 dV2 TV1 DNA 11 pCMV 140 dV2 SF162 DNA + pCMV pCMV 140 dV2 SF162 DNA + pCMV pCMV 140 dV2 SF162 DNA + pCMV Protein + MF59C 140 dV1/V2 TV1 DNA 140 dV1/V2 TV1 DNA 140 dV1/V2 TV1 DNA

[0397] TABLE-US-00007 TABLE G Bleed: 0 1 2 3 4 5 6 7 8 9 10 Week: -3 4 6 8 12 16 18 20 24 28 TBD Sample: Clotted Clotted Clotted Clotted Clotted Clotted Clotted Clotted Clotted Bld. Clotted Bld. Clotted Bld. Bld. Bld. Bld. Bld. Bld. Bld. Bld. Bld. for Serum for Serum for Serum for Serum for Serum for Serum for Serum for Serum for Serum for Serum for Serum Volume: 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each Method: AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV CP

Example 10

Mice Immunization Studies with Gag and Pol Constructs

[0398] Cellular and Humoral immune responses were evaluated in mice (essentially as described in Example 4) for the following constructs: Gag, GagProtease(+FS) (GP1, protease codon optimized and inactivation of INS; GP2, protease only inactivation of INS), GagPol.DELTA.integrase with frameshift (gagFSpol), and GagPol.DELTA.integrase in-frame (GagPol) (see FIG. 63). Versions of GagPol.DELTA.integrase in-frame were also designed with attenuated (GagPolAtt) or non-functional Protease (GagPolIna).

[0399] In vitro expression data showed comparable expression of p55Gag and p66RT using Gag alone, GagProtease(+FS), GagFSpol and GagPolIna. Constructs with fully functional or attenuated protease (GagPol or GagPolAtt) were less efficient in expression of p55Gag and p66RT, possibly due to cytotoxic effects of protease.

[0400] DNA immunization of mice using Gag vs. GP1 and GP2 in pCMV vectors was performed intramuscularly in the tibialis anterior. Mice were immunized at the start of the study (0 week) and 4 weeks later. Bleeds were performed at 0, 4, and 6 weeks. DNA doses used were as follows: 20 .mu.g, 2 .mu.g, 0.2 .mu.g, and 0.02 .mu.g.

[0401] DNA immunization of mice using Gag vs. gagFSpol in pCMV vectors was performed intramuscularly in the tibialis anterior. Mice were immunized at the start of the study (0 week) and challenged 4 weeks later with recombinant vaccinia virus encoding Gag (rVVgag). Bleeds were performed at 0 and 4 weeks. DNA doses used were as follows: 20 .mu.g, 2 .mu.g, 0.2 .mu.g, and 0.02 .mu.g.

[0402] DNA immunization of mice using Gag vs. gagFSpol and gagpol in pCMV vectors was performed intramuscularly in the tibialis anterior. Mice were immunized at the start of the study (0 week) and challenged 4 weeks later with recombinant vaccinia virus encoding Gag (rVVgag). Bleeds were performed at 0 and 4 weeks. DNA doses used were as follows: 2 .mu.g, 0.2 .mu.g, 0.02 .mu.g, and 0.002 .mu.g.

[0403] Cellular immune responses against Gag were comparable for all tested variants, for example, Gag, GagProtease, gagFSpol and GagPolIna all had comparable potencies.

[0404] Humoral immune responses to Gag were also comparable with the exception of GP2 and especially GP1. Humoral immune responses were weaker in constructs comprising functional or attenuated proteases which may be due to less efficient secretion of p55Gag caused by overactive protease.

[0405] In vitro and in vivo experiments, performed in support of the present invention, suggest that the expression and immunogenicity of Gag was comparable with all constructs. Exceptions were GagPol in-frame with fully functional or attenuated protease. This may be the result of cytotoxic effects of protease. The immune response in mice correlated with relative levels of expression in vitro.

Example 11

Protein Expression, Immunogenicity, and Generation of Neutralizing Antibodies Using Type C Derived Envelope Polypeptides

[0406] Envelope (Env) vaccines derived from the subtype C primary isolate, TV1, recovered from a South African individual, were tested in rabbits as follows. Gene cassettes were designed to express the gp120 (surface antigen), gp140 (surface antigen plus ectodomain of transmembrane protein, gp41), and full-length (gp120 plus gp41) gp160 forms of the HIV-1 envelope polyprotein with and without deletions of the variable loop regions, V2 and V1V2. All of the genes were sequence-modified to enhance expression of the encoded Env glycoproteins in a Rev-independent fashion and they were subsequently cloned into pCMV-based plasmid vectors for DNA vaccine and protein production applications as described above. The sequences were codon optimized as described herein. Briefly, all the modified envelope genes were cloned into the Chiron pCMVlink plasmid vector, preferably into EcoRI/XhoI sites.

[0407] A. Protein Expression

[0408] Full-length (gp160), truncated gp140 (Env ectodomain only) and gp120 native versions of the TV1 Env antigen were produced from the expression cassettes described herein. The gp140 encoding sequences were transiently transfected into 293T cells. The expression levels of the gene products were evaluated by an in-house antigen capture ELISA. Envelope genes constructed from the native sequences of TV001c8.2, TV001c8.5 and TV002c12.1 expressed the correct proteins in vitro, with gp140TV001c8.2 exhibiting the highest level of expression. In addition, the Env protein expressed from the TV1-derived clone 8.2 was found to bind the CD4 receptor protein indicating that this feature of the expressed protein is maintained in a functional conformation. The receptor binding properties/functionality of the expressed TV1 gp160 protein result was also confirmed by a cell-fusion assay.

[0409] Total expression increased approximately 10-fold for synthetic gp140 constructs compared with the native gp140 gene cassettes. Both the modified gp120 and gp140 variants secreted high amounts of protein in the supernatant. In addition, the V2 and V1V2 deleted forms of gp140 expressed approximately 2-fold more protein than the intact gp140. Overall, the expression levels of synthetic gp140 gene variants increased 10 to 26-fold compared with the gp140 gene with native sequences.

[0410] In sum, each synthetic construct tested showed more than 10-fold increased levels of expression relative to those using the native coding sequences. Moreover, all expressed proteins were of the expected molecular weights and were shown to bind CD4. Stable CHO cell lines were derived and small-scale protein purification methods were used to produce small quantities of each of the undeleted and V-deleted oligomeric forms (o-gp140) of these proteins for vaccine studies.

[0411] B. Neutralization Properties of TV001 and TV002 Viral Isolates

[0412] The transient expression experiment showed that the envelope genes derived from the TV001 and TV002 virus isolates expressed the desired protein products. Relative neutralization sensitivities of these two viral strains using sera from 18 infected South African individuals (subtypes B and C) were as follows. At a 1:10 serum dilution, the TV2 strain was neutralized by 18 of 18 sera; at 1:50, 16 of 18; at 1:250, 15/18. In comparison, the TV1 isolate was neutralized by 15 of 18 at 1:10; only 6 of 18 at 1:50; and none of the specimens at 1:250. In addition, the TV001 patient serum showed neutralization activity against the TV002 isolate at all dilutions tested. In contrast, the TV002 showed neutralization of TV001 only at the 1:10 serum dilution. These results suggest that TV001 isolate is capable of inducing a broader and more potent neutralizing antibody response in its infected host than TV002.

[0413] C. Immunogenicity of the Modified TV1 Env DNA and Protein Antigens in Rabbit Studies

[0414] TV1 Env DNA (comprising the synthetic expression cassettes) and protein vaccines were administrated as shown in the following Table H. TABLE-US-00008 TABLE H Groups Plasmid DNA (0, 4, and 20 wks) Protein boost (20 wks) 1 pCMVgp160.TV1 o-gp140.TV1 2 pCMVgp160dV2.TV1 o-gp140dV2.TV1 3 pCMVgp160dV1V2.TV1 o-gp140dV1V2.TV1 4 pCMVgp140.TV1 o-gp140.TV1 5 pCMVgp140dV2.TV1 o-gp140dV2.TV1 6 pCMVgp140dV1V2.TV1 o-gp140dV1V2.TV1 7 pCMVgp140dV2.SF162 o-gp140dV2.SF162

[0415] Seven groups of 4 rabbits per group were immunized with the designated plasmid DNA and oligomeric Env protein antigens. Three doses of DNA, 1 mg of DNA per animal per immunization, were administrated intramuscularly by needle injection followed by electroporation on weeks 0, 4, and 20 weeks. A single dose of 100 ug of Env protein in MF59 adjuvant also was given intramuscularly in a separate site at 20 weeks.

[0416] The DNA immunization used subtype C sequence-modified genes (TV1)--gp160, gp160dV2, gp160dV1V2, gp140, gp140dV2 and gp140dV1V2--as well as a subtype B SF162 sequence modified gp140dV2. DNA immunizations were performed at 0, 4, and 20 weeks by needle injection by the intramuscular route using electroporation to facilitate transfection of the muscle cells and of resident antigen presenting cells.

[0417] A single Env protein booster (in MF59 adjuvant) was given at 20 weeks by intramuscular injection at a separate site. Antibody titers were evaluated by ELISA following each successive immunization. Serum specimens were collected at 0, 4, 6, 8, 12, 22, and 24 weeks. Serum antibody titers were measured on ELISA. 96-well plates were coated with a protein in a concentration of 1 ug/ml. Serum samples were diluted serially 3-fold. Goat anti-rabbit peroxidase conjugate (1:20,000) was used for detection. TMB was used as the substrate, and the antibody titers were read at 0.6 OD at 450 nm.

[0418] Neutralizing antibody responses against PBMC-grown R5 HIV-1 strains were monitored in the sera collected from the immunized rabbits using two different assays in two different laboratories, the 5.25 reporter cell-line based assay at Chiron and the PBMC-based assay of David Montefiori at Duke University. Results are shown in FIGS. 66, 67, and 68. The Chiron assay was conducted essentially as follows. Neutralizing antibody responses against the PBMC-grown subtype C TV001 and TV002 strains were measured using an in-house reporter cell line assay that uses the 5.25 cell line. This cell has CD4, CCR5, CXCR4 and BONZO receptor/co-receptors on its cell membrane. The parental CEM cell line was derived from a 4-year-old Caucasian female with acute lymphoblastic leukemia, which was fused with the human B cell line 721.174, creating CEMx174. LTR-GFP was transfected into the cells after the CCR5 gene (about 1.1 kb) was cloned into the BamH-I (5') and Sal-I (3') of the pBABE puro retroviral vector, and subsequently introduced into the CEMx174. The green fluorescence protein (GFP) of the cells was detected by flow cytometer (FACScan). For the virus neutralization assay, 50 ul of titrated virus and 50 ul of diluted immune or pre-immune serum were incubated at room temperature for one hour. This mixture was added into wells with 10.sup.4/ml cells plated in a 24 well plate, and incubated at 37.degree. C. for 5 to 7 days. The cells were then fixed with 2% of formaldehyde after washing with PBS. Fifteen thousand events (cells) were collected for each sample on a Becton Dickinson FACScan using Cellquest software. The data presented were the mean of the triplicate wells. The percent neutralization was calculated compared to the virus control using the following equation: % virus Inhibition=(virus control-experimental)/(virus control-cell control).times.100. Any virus inhibition observed in the pre-bleed has been subtracted for each individual animal. Values >50% are considered positive and are highlighted in gray.

[0419] In FIG. 67, the "#" indicates that animals had high levels of virus inhibition in pre-bleed serum (>20% virus inhibition) that impacted the magnitude of the observed inhibition and in some cases, our ability to score the serum as a positive or negative for the presence of significant neutralizing antibody activity (<50% inhibition).

[0420] For the data presented in FIG. 68, serum samples were collected after a single protein boost (post-third) were screened in triplicate at a 1:8 dilution with virus (1:24 after addition of cells). Values shown are the % reduction in p24 synthesis relative to that in the corresponding pre-bleed control samples. Zero values indicate no or negative values were measured. NV, not valid due to virus inhibition in pre-immune serum. Neutralization was considered positive when p24 was reduced by at least 80%; these samples are highlighted in dark gray. Sample with lighter gray shading showed at least a 50% reduction in p24 synthesis.

[0421] FIG. 64 shows the ELISA data when plates were coated with the monomeric gp120.TV1 protein. This protein is homologous to the subtype C genes used for the immunization. All immunization groups produced high antibody titers after the second DNA immunization. The groups immunized with gp140 forms of DNA have relatively higher geometric mean antibody titers as compared to the groups using gp160 forms after both first and second DNA immunizations. Both the gp140.TV1 and gp140dV1V2.TV1 genes produced high antibody titers at about 10.sup.4 at two weeks post second DNA; the gp140dV2.TV1 plasmid yielded the highest titers of antibodies (>10.sup.4) at this time point and all others. The binding antibody titers to the gp120.TV1 protein were higher for the group immunized with the homologous gp140dV2.TV1 genes than that with the heterologous gp140dV2.5F162 gene which showed titers of about 10.sup.3. All the groups, showed some decline in antibody titers by 8 weeks post the second DNA immunization. Following the DNA plus protein booster at 20 weeks, all groups reached titers above that previously observed after the second DNA immunization (0.5-1.0 log increases were observed). After the protein boost, all animals receiving the o-gp140dV2.TV1 protein whether primed by the gp140dV2.TV1 or gp160dV2.TV1 DNA, showed the highest Ab titers.

[0422] Binding antibody titers were also measured using ELISA plates coated with either oligomeric subtype C o-gp140dV2.TV1 or subtype B o-gp140dV2.5F162 proteins (FIG. 65). For all the TV1 Env immunized groups, the antibody titers measured using the oligomeric protein, o-gp140dV2.TV1 were higher than those measured using the monomeric (non-V2-deleted) protein, gp120.TV1. In fact, for these groups, the titers observed with the heterologous subtype B o-gp140dV2.5F162 protein were comparable to or greater than those measured with the subtype C TV1 gp120. Nevertheless, all groups immunized with subtype C immunogens showed higher titers binding to the subtype C o-gp140dV2.TV1 protein than to the subtype B protein gp140dV2.5F162. Conversely, the group immunized with the gp140dV2.5F162 immunogen showed higher antibody titers with the oligomeric subtype B protein relative its subtype C counterpart. Overall, all three assays demonstrated that high antibody cross-reactive antibodies were generated by the subtype CTV1-based DNA and protein immunogens.

[0423] The results indicate that the subtype CTV1-derived Env DNA and protein antigens are immunogenic inducing high titers of antibodies in immunized rabbits and substantial evidence of neutralizing antibodies against both subtype B and subtype C R5 virus strains. In particular, the gp140dV2.TV1 antigens have induced consistent neutralizing responses against the subtype B SF162EnvDV2 and subtype C TV2 strains. Thus, TV1-based Env DNA and protein-based antigens are immunogenic and induce high titer antibody responses reactive with both subtype C and subtype B HIV-1 Env antigens. Neutralizing antibody responses against the neutralization sensitive subtype B R5 HIV-1.sub.SF162DV2 strain were observed in some groups after only two DNA immunizations. Following a single booster immunization with Env protein, the majority of rabbits in groups that received V2-deleted forms of the TV1 Env showed neutralization activity against the closely related subtype C TV2 primary strain.

Example 12

Immunological Responses in Rhesus Macaques

[0424] Cellular and humoral immune responses were evaluated in three groups of rhesus macaques (each group was made up of four animals) in an immunization study structured as shown in Table I. The route of administration for the immunizing composition was electroporation in each case. Antibody titers are shown in Table I for two weeks post-second immunization. TABLE-US-00009 TABLE I Formulation of Immunizing Group Composition* Animal # Titer 1 pCMVgag (3.5 mg) + pCMVenv A 3325 (2.0 mg) B 4000 C (previously 1838 immunized with HCV core ISCOMS, rVVC core E1) D (previously 1850 immunized with HCV core ISCOMS, rVVC core E1) 2 pCMVgag (3.5 mg) + pCMVpol A (previously 525 (4.2 mg) immunized with HCV core ISCOMS, rVVC core E1, p55gag.sub.LAI(VLP)) B 5313 C 6450 D 5713 3 pCMVgag-pol A (previously 0 (5.0 mg) immunized with HCV core ISCOMS, rVVC core E1, pCMVgagSF2) B (previously 1063 immunized with rVVC/E1, pCMV Epo-Epi, HIV/HCV-VLP, pCMVgagSF2, pUCgp120 SF2) C 513 D (previously 713 immunized with rVVC/E1, HIV/HCV-VLP) *pCMVgag = pCMVKm2.GagMod Type C Botswana pCMVenv = pCMVLink.gp140env.dV2.TV1 (Type C) pCMVpol = pCMVKm2.p2Pol.mut.Ina Type C Botswana pCMVgag-pol = pCMVKm2.gagCpol.mut.Ina Type C Botswana

[0425] Pre-immune sera were obtained at week 0 before the first immunization. The first immunization was given at week 0. The second immunization was given at week 4. The first bleed was performed at 2 weeks post-second immunization (i.e., at week 6). A third immunization will be given at week 8 and a fourth at week 16. Animals 2A, 3A, 3B and 3D had been vaccinated previously (approximately 4 years or more) with gag plasmid DNA or gag VLP (subtype B).

[0426] Bulk CTL, .sup.51Cr-release assays, and flow cell cytometry methods were used to obtain the data in Tables J and K. Reagents used for detecting gag- and pol-specific T-cells were (i) synthetic, overlapping peptides spanning "gagCpol" antigen (n=377), typically the peptides were pools of 15-mers with overlap by 11, the pools were as follows, pool 1, n=1-82, pool 2, n=83-164, pool 3, n=165-271, pool 4, n=272-377, accordingly pools 1 and 2 are "gag"-specific, and pools 3 and 4 are "pol"-specific, and (ii) recombinant vaccinia virus (rVV), for example, rVVgag965, rVVp2Pol975 (contains p2p7gag975), and VV.sub.wrparent.

[0427] Gag-specific IFN.gamma.+CD8+ T-cells, Gag-specific IFN.gamma.+CD4+ T-cells, Pol-specific IFN.gamma.+CD8+ T-cells, and Pol-specific IFN.gamma.+CD4+ T-cells in blood were determined for each animal described in Table I above, post second immunization. The results are presented in Tables J and K. It is possible that some of the pol-specific activity shown in Table K was directed against p2p7gag. TABLE-US-00010 TABLE J Gag Assay Results Gag Specific CD4+ Gag Specific CD8+ Responses Responses Group/ Immunizing LPA(SI) Flow CTL Flow Animal Composition p55 Pool 1 Pool 2 IFNg+ Pool 1 Pool 2 IFNg+ 1A pCMVgag 3.3 5.9 3.8 496 minus minus 225 pCMVenv 1B pCMVgag 11.8 4.4 1.5 786 minus minus 160 pCMVenv 1C pCMVgag 5.7 1.1 2.4 361 plus plus 715 pCMVenv 1D pCMVgag 6.5 3.1 1.6 500 plus ? 596 pCMVenv 2A pCMVgag 4.8 4.8 1.6 405 plus minus 1136 pCMVpol 2B pCMVgag 12.5 6.8 3.3 1288 plus minus 2644 pCMVpol 2C pCMVgag 6 3.8 2.1 776 minus minus 0 pCMVpol 2D pCMVgag 18.9 13.5 5.4 1351 minus minus 145 pCMVpol 3A pCMV 12.2 7 1.5 560 plus plus 3595 gagpol 3B pCMV 2.7 5.6 1.3 508 plus ? 3256 gagpol 3C pCMV 11.6 5 1.2 289 minus ? 617 gagpol 3D pCMV 1.5 1.2 1.4 120 minus minus 277 gagpol ? = might be positive on rVVp2Pol.

[0428] TABLE-US-00011 TABLE K Pol Assay Results Pol Specific CD4+ Pol Specific CD8+ Immu- Responses Responses nizing LPA(SI) CTL Group/ Com- Pool Pool Flow Pool Pool Flow Animal position 3 4 IFNg+ 3 4 IFNg+ 1A pCMVgag 1 1.2 0 minus minus 0 pCMVenv 1B pCMVgag 1 1 0 minus minus 0 pCMVenv 1C pCMVgag 1 1.1 0 minus minus 0 pCMVenv 1D pCMVgag 1.2 1.3 0 minus minus 262 pCMVenv 2A pCMVgag 1.1 0.9 92 minus minus 459 pCMVpol 2B pCMVgag 2.5 1.8 107 minus minus 838 pCMVpol 2C pCMVgag 1.2 1.1 52 plus minus 580 pCMVpol 2D pCMVgag 2.5 2.7 113 plus plus 5084 pCMVpol 3A pCMV 2.7 2.4 498 minus minus 3631 gagpol 3B pCMV 1.1 1 299 minus minus 1346 gagpol 3C pCMV 2.1 1.4 369 minus minus 399 gagpol 3D pCMV 1.3 1.8 75 minus minus 510 gagpol

[0429] These results support that the constructs of the present invention are capable of generating specific cellular and humoral responses against the selected HIV-polypeptide antigens.

[0430] Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.

Sequence CWU 1

1

68 1 9781 DNA Artificial Sequence Description of Artificial Sequence 8_5_TV1_C.ZA 1 tggaagggtt aatttactcc aagaaaaggc aagaaatcct tgatttgtgg gtctatcaca 60 cacaaggctt cttccctgat tggcaaaact acacaccggg gccaggggtc agatatccac 120 tgacctttgg atggtgctac aagctagtgc cagttgaccc aggggaggtg gaagaggcca 180 acggaggaga agacaactgt ttgctacacc ctatgagcca acatggagca gaggatgaag 240 atagagaagt attaaagtgg aagtttgaca gcctcctagc acgcagacac atggcccgcg 300 agctacatcc ggagtattac aaagactgct gacacagaag ggactttccg cctgggactt 360 tccactgggg cgttccggga ggtgtggtct gggcgggact tgggagtggt caaccctcag 420 atgctgcata taagcagctg cttttcgcct gtactgggtc tctctcggta gaccagatct 480 gagcctggga gccctctggc tatctaggga acccactgct taagcctcaa taaagcttgc 540 cttgagtgct ttaagtagtg tgtgcccatc tgttgtgtga ctctggtaac tagagatccc 600 tcagaccctt tgtggtagtg tggaaaatct ctagcagtgg cgcccgaaca gggaccagaa 660 agtgaaagtg agaccagagg agatctctcg acgcaggact cggcttgctg aagtgcacac 720 ggcaagaggc gagaggggcg gctggtgagt acgccaattt tacttgacta gcggaggcta 780 gaaggagaga gatgggtgcg agagcgtcaa tattaagcgg cggaaaatta gataaatggg 840 aaagaattag gttaaggcca gggggaaaga aacattatat gttaaaacat ctagtatggg 900 caagcaggga gctggaaaga tttgcactta accctggcct gttagaaaca tcagaaggct 960 gtaaacaaat aataaaacag ctacaaccag ctcttcagac aggaacagag gaacttagat 1020 cattattcaa cacagtagca actctctatt gtgtacataa agggatagag gtacgagaca 1080 ccaaggaagc cttagacaag atagaggaag aacaaaacaa atgtcagcaa aaagcacaac 1140 aggcaaaagc agctgacgaa aaggtcagtc aaaattatcc tatagtacag aatgcccaag 1200 ggcaaatggt acaccaagct atatcaccta gaacattgaa tgcatggata aaagtaatag 1260 aggaaaaggc tttcaatcca gaggaaatac ccatgtttac agcattatca gaaggagcca 1320 ccccacaaga tttaaacaca atgttaaata cagtgggggg acatcaagca gccatgcaaa 1380 tgttaaaaga taccatcaat gaggaggctg cagaatggga taggacacat ccagtacatg 1440 cagggcctgt tgcaccaggc cagatgagag aaccaagggg aagtgacata gcaggaacta 1500 ctagtaccct tcaggaacaa atagcatgga tgacaagtaa tccacctatt ccagtagaag 1560 acatctataa aagatggata attctggggt taaataaaat agtaagaatg tatagccctg 1620 ttagcatttt ggacataaaa caagggccaa aagaaccctt tagagactat gtagaccggt 1680 tctttaaaac cttaagagct gaacaagcta cacaagatgt aaagaattgg atgacagaca 1740 ccttgttggt ccaaaatgcg aacccagatt gtaagaccat tttaagagca ttaggaccag 1800 gggcctcatt agaagaaatg atgacagcat gtcagggagt gggaggacct agccataaag 1860 caagagtgtt ggctgaggca atgagccaag caaacagtaa catactagtg cagagaagca 1920 attttaaagg ctctaacaga attattaaat gtttcaactg tggcaaagta gggcacatag 1980 ccagaaattg cagggcccct aggaaaaagg gctgttggaa atgtggacag gaaggacacc 2040 aaatgaaaga ctgtactgag aggcaggcta attttttagg gaaaatttgg ccttcccaca 2100 aggggaggcc agggaatttc ctccagaaca gaccagagcc aacagcccca ccagcagaac 2160 caacagcccc accagcagag agcttcaggt tcgaggagac aacccccgtg ccgaggaagg 2220 agaaagagag ggaaccttta acttccctca aatcactctt tggcagcgac cccttgtctc 2280 aataaaagta gagggccaga taaaggaggc tctcttagac acaggagcag atgatacagt 2340 attagaagaa atagatttgc cagggaaatg gaaaccaaaa atgatagggg gaattggagg 2400 ttttatcaaa gtaagacagt atgatcaaat acttatagaa atttgtggaa aaaaggctat 2460 aggtacagta ttagtagggc ctacaccagt caacataatt ggaagaaatc tgttaactca 2520 gcttggatgc acactaaatt ttccaattag tcctattgaa actgtaccag taaaattaaa 2580 accaggaatg gatggcccaa aggtcaaaca atggccattg acagaagaaa aaataaaagc 2640 attaacagca atttgtgagg aaatggagaa ggaaggaaaa attacaaaaa ttgggcctga 2700 taatccatat aacactccag tatttgccat aaaaaagaag gacagtacta agtggagaaa 2760 attagtagat ttcagggaac tcaataaaag aactcaagac ttttgggaag ttcaattagg 2820 aataccacac ccagcaggat taaaaaagaa aaaatcagtg acagtgctag atgtggggga 2880 tgcatatttt tcagttcctt tagatgaaag cttcaggaaa tatactgcat tcaccatacc 2940 tagtataaac aatgaaacac cagggattag atatcaatat aatgtgctgc cacagggatg 3000 gaaaggatca ccagcaatat tccagagtag catgacaaaa atcttagagc ccttcagagc 3060 aaaaaatcca gacatagtta tctatcaata tatggatgac ttgtatgtag gatctgactt 3120 agaaataggg caacatagag caaaaataga agagttaagg gaacatttat tgaaatgggg 3180 atttacaaca ccagacaaga aacatcaaaa agaaccccca tttctttgga tggggtatga 3240 actccatcct gacaaatgga cagtacaacc tatactgctg ccagaaaagg atagttggac 3300 tgtcaatgat atacagaagt tagtgggaaa attaaactgg gcaagtcaga tttacccagg 3360 gattaaagta aggcaactct gtaaactcct caggggggcc aaagcactaa cagacatagt 3420 accactaact gaagaagcag aattagaatt ggcagagaac agggaaattt taagagaacc 3480 agtacatgga gtatattatg atccatcaaa agacttgata gctgaaatac agaaacaggg 3540 gcatgaacaa tggacatatc aaatttatca agaaccattt aaaaatctga aaacagggaa 3600 gtatgcaaaa atgaggacta cccacactaa tgatgtaaaa cagttaacag aggcagtgca 3660 aaaaatagcc atggaaagca tagtaatatg gggaaagact cctaaattta gactacccat 3720 ccaaaaagaa acatgggaga catggtggac agactattgg caagccacct ggatccctga 3780 gtgggagttt gttaataccc ctcccctagt aaaattatgg taccaactag aaaaagatcc 3840 catagcagga gtagaaactt tctatgtaga tggagcaact aatagggaag ctaaaatagg 3900 aaaagcaggg tatgttactg acagaggaag gcagaaaatt gttactctaa ctaacacaac 3960 aaatcagaag actgagttac aagcaattca gctagctctg caggattcag gatcagaagt 4020 aaacatagta acagactcac agtatgcatt aggaatcatt caagcacaac cagataagag 4080 tgactcagag atatttaacc aaataataga acagttaata aacaaggaaa gaatctacct 4140 gtcatgggta ccagcacata aaggaattgg gggaaatgaa caagtagata aattagtaag 4200 taagggaatt aggaaagtgt tgtttctaga tggaatagat aaagctcaag aagagcatga 4260 aaggtaccac agcaattgga gagcaatggc taatgagttt aatctgccac ccatagtagc 4320 aaaagaaata gtagctagct gtgataaatg tcagctaaaa ggggaagcca tacatggaca 4380 agtcgactgt agtccaggga tatggcaatt agattgtacc catttagagg gaaaaatcat 4440 cctggtagca gtccatgtag ctagtggcta catggaagca gaggttatcc cagcagaaac 4500 aggacaagaa acagcatatt ttatattaaa attagcagga agatggccag tcaaagtaat 4560 acatacagac aatggcagta attttaccag tactgcagtt aaggcagcct gttggtgggc 4620 aggtatccaa caggaatttg gaattcccta caatccccaa agtcagggag tggtagaatc 4680 catgaataaa gaattaaaga aaataatagg acaagtaaga gatcaagctg agcaccttaa 4740 gacagcagta caaatggcag tattcattca caattttaaa agaaaagggg gaattggggg 4800 gtacagtgca ggggaaagaa taatagacat aatagcaaca gacatacaaa ctaaagaatt 4860 acaaaaacaa attataagaa ttcaaaattt tcgggtttat tacagagaca gcagagaccc 4920 tatttggaaa ggaccagccg aactactctg gaaaggtgaa ggggtagtag taatagaaga 4980 taaaggtgac ataaaggtag taccaaggag gaaagcaaaa atcattagag attatggaaa 5040 acagatggca ggtgctgatt gtgtggcagg tggacaggat gaagattaga gcatggaata 5100 gtttagtaaa gcaccatatg tatatatcaa ggagagctag tggatgggtc tacagacatc 5160 attttgaaag cagacatcca aaagtaagtt cagaagtaca tatcccatta ggggatgcta 5220 gattagtaat aaaaacatat tggggtttgc agacaggaga aagagattgg catttgggtc 5280 atggagtctc catagaatgg agactgagag aatacagcac acaagtagac cctgacctgg 5340 cagaccagct aattcacatg cattattttg attgttttac agaatctgcc ataagacaag 5400 ccatattagg acacatagtt tttcctaggt gtgactatca agcaggacat aagaaggtag 5460 gatctctgca atacttggca ctgacagcat tgataaaacc aaaaaagaga aagccacctc 5520 tgcctagtgt tagaaaatta gtagaggata gatggaacga cccccagaag accaggggcc 5580 gcagagggaa ccatacaatg aatggacact agagattcta gaagaactca agcaggaagc 5640 tgtcagacac tttcctagac catggctcca tagcttagga caatatatct atgaaaccta 5700 tggggatact tggacgggag ttgaagctat aataagagta ctgcaacaac tactgttcat 5760 tcatttcaga attggatgcc aacatagcag aataggcatc ttgcgacaga gaagagcaag 5820 aaatggagcc agtagatcct aaactaaagc cctggaacca tccaggaagc caacctaaaa 5880 cagcttgtaa taattgcttt tgcaaacact gtagctatca ttgtctagtt tgctttcaga 5940 caaaaggttt aggcatttcc tatggcagga agaagcggag acagcgacga agcgctcctc 6000 caagtggtga agatcatcaa aatcctctat caaagcagta agtacacata gtagatgtaa 6060 tggtaagttt aagtttattt aaaggagtag attatagatt aggagtagga gcattgatag 6120 tagcactaat catagcaata atagtgtgga ccatagcata tatagaatat aggaaattgg 6180 taagacaaaa gaaaatagac tggttaatta aaagaattag ggaaagagca gaagacagtg 6240 gcaatgagag tgatggggac acagaagaat tgtcaacaat ggtggatatg gggcatctta 6300 ggcttctgga tgctaatgat ttgtaacacg gaggacttgt gggtcacagt ctactatggg 6360 gtacctgtgt ggagagaagc aaaaactact ctattctgtg catcagatgc taaagcatat 6420 gagacagaag tgcataatgt ctgggctaca catgcttgtg tacccacaga ccccaaccca 6480 caagaaatag ttttgggaaa tgtaacagaa aattttaata tgtggaaaaa taacatggca 6540 gatcagatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc atgtgtaaag 6600 ttgaccccac tctgtgtcac tttaaactgt acagatacaa atgttacagg taatagaact 6660 gttacaggta atacaaatga taccaatatt gcaaatgcta catataagta tgaagaaatg 6720 aaaaattgct ctttcaatgc aaccacagaa ttaagagata agaaacataa agagtatgca 6780 ctcttttata aacttgatat agtaccactt aatgaaaata gtaacaactt tacatataga 6840 ttaataaatt gcaatacctc aaccataaca caagcctgtc caaaggtctc ttttgacccg 6900 attcctatac attactgtgc tccagctgat tatgcgattc taaagtgtaa taataagaca 6960 ttcaatggga caggaccatg ttataatgtc agcacagtac aatgtacaca tggaattaag 7020 ccagtggtat caactcaact actgttaaat ggtagtctag cagaagaagg gataataatt 7080 agatctgaaa atttgacaga gaataccaaa acaataatag tacatcttaa tgaatctgta 7140 gagattaatt gtacaaggcc caacaataat acaaggaaaa gtgtaaggat aggaccagga 7200 caagcattct atgcaacaaa tgacgtaata ggaaacataa gacaagcaca ttgtaacatt 7260 agtacagata gatggaataa aactttacaa caggtaatga aaaaattagg agagcatttc 7320 cctaataaaa caataaaatt tgaaccacat gcaggagggg atctagaaat tacaatgcat 7380 agctttaatt gtagaggaga atttttctat tgcaatacat caaacctgtt taatagtaca 7440 tactacccta agaatggtac atacaaatac aatggtaatt caagcttacc catcacactc 7500 caatgcaaaa taaaacaaat tgtacgcatg tggcaagggg taggacaagc aatgtatgcc 7560 cctcccattg caggaaacat aacatgtaga tcaaacatca caggaatact attgacacgt 7620 gatgggggat ttaacaacac aaacaacgac acagaggaga cattcagacc tggaggagga 7680 gatatgaggg ataactggag aagtgaatta tataaatata aagtggtaga aattaagcca 7740 ttgggaatag cacccactaa ggcaaaaaga agagtggtgc agagaaaaaa aagagcagtg 7800 ggaataggag ctgtgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 7860 tcaataacgc tgacggtaca ggccagacaa ctgttgtctg gtatagtgca acagcaaagc 7920 aatttgctga aggctataga ggcgcaacag catatgttgc aactcacagt ctggggcatt 7980 aagcagctcc aggcgagagt cctggctata gaaagatacc taaaggatca acagctccta 8040 gggatttggg gctgctctgg aagactcatc tgcaccactg ctgtgccttg gaactccagt 8100 tggagtaata aatctgaagc agatatttgg gataacatga cttggatgca gtgggataga 8160 gaaattaata attacacaga aacaatattc aggttgcttg aagactcgca aaaccagcag 8220 gaaaagaatg aaaaagattt attagaattg gacaagtgga ataatctgtg gaattggttt 8280 gacatatcaa actggctgtg gtatataaaa atattcataa tgatagtagg aggcttgata 8340 ggtttaagaa taatttttgc tgtgctctct atagtgaata gagttaggca gggatactca 8400 cctttgtcat ttcagaccct taccccaagc ccgaggggac tcgacaggct cggaggaatc 8460 gaagaagaag gtggagagca agacagagac agatccatac gattggtgag cggattcttg 8520 tcgcttgcct gggacgatct gcggagcctg tgcctcttca gctaccaccg cttgagagac 8580 ttcatattaa ttgcagtgag ggcagtggaa cttctgggac acagcagtct caggggacta 8640 cagagggggt gggagatcct taagtatctg ggaagtcttg tgcagtattg gggtctagag 8700 ctaaaaaaga gtgctattag tccgcttgat accatagcaa tagcagtagc tgaaggaaca 8760 gataggatta tagaattggt acaaagaatt tgtagagcta tcctcaacat acctaggaga 8820 ataagacagg gctttgaagc agctttgcta taaaatggga ggcaagtggt caaaacgcag 8880 catagttgga tggcctgcag taagagaaag aatgagaaga actgagccag cagcagaggg 8940 agtaggagca gcgtctcaag acttagatag acatggggca cttacaagca gcaacacacc 9000 tgctactaat gaagcttgtg cctggctgca agcacaagag gaggacggag atgtaggctt 9060 tccagtcaga cctcaggtac ctttaagacc aatgacttat aagagtgcag tagatctcag 9120 cttcttttta aaagaaaagg ggggactgga agggttaatt tactctagga aaaggcaaga 9180 aatccttgat ttgtgggtct ataacacaca aggcttcttc cctgattggc aaaactacac 9240 atcggggcca ggggtccgat tcccactgac ctttggatgg tgcttcaagc tagtaccagt 9300 tgacccaagg gaggtgaaag aggccaatga aggagaagac aactgtttgc tacaccctat 9360 gagccaacat ggagcagagg atgaagatag agaagtatta aagtggaagt ttgacagcct 9420 tctagcacac agacacatgg cccgcgagct acatccggag tattacaaag actgctgaca 9480 cagaagggac tttccgcctg ggactttcca ctggggcgtt ccgggaggtg tggtctgggc 9540 gggacttggg agtggtcacc ctcagatgct gcatataagc agctgctttt cgcttgtact 9600 gggtctctct cggtagacca gatctgagcc tgggagctct ctggctatct agggaaccca 9660 ctgcttaggc ctcaataaag cttgccttga gtgctctaag tagtgtgtgc ccatctgttg 9720 tgtgactctg gtaactagag atccctcaga ccctttgtgg tagtgtggaa aatctctagc 9780 a 9781 2 842 PRT Artificial Sequence Description of Artificial Sequence SF162 2 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Ala Val Glu Lys Leu Trp Val Thr Val 20 25 30 Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys 35 40 45 Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala 50 55 60 Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Ile Val Leu 65 70 75 80 Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu 85 90 95 Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro 100 105 110 Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Leu 115 120 125 Lys Asn Ala Thr Asn Thr Lys Ser Ser Asn Trp Lys Glu Met Asp Arg 130 135 140 Gly Glu Ile Lys Asn Cys Ser Phe Lys Val Thr Thr Ser Ile Arg Asn 145 150 155 160 Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro 165 170 175 Ile Asp Asn Asp Asn Thr Ser Tyr Lys Leu Ile Asn Cys Asn Thr Ser 180 185 190 Val Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile 195 200 205 His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys 210 215 220 Lys Phe Asn Gly Ser Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cys 225 230 235 240 Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly 245 250 255 Ser Leu Ala Glu Glu Gly Val Val Ile Arg Ser Glu Asn Phe Thr Asp 260 265 270 Asn Ala Lys Thr Ile Ile Val Gln Leu Lys Glu Ser Val Glu Ile Asn 275 280 285 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Thr Ile Gly Pro 290 295 300 Gly Arg Ala Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln 305 310 315 320 Ala His Cys Asn Ile Ser Gly Glu Lys Trp Asn Asn Thr Leu Lys Gln 325 330 335 Ile Val Thr Lys Leu Gln Ala Gln Phe Gly Asn Lys Thr Ile Val Phe 340 345 350 Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn 355 360 365 Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe Asn Ser 370 375 380 Thr Trp Asn Asn Thr Ile Gly Pro Asn Asn Thr Asn Gly Thr Ile Thr 385 390 395 400 Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Arg Trp Gln Glu Val Gly 405 410 415 Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Arg Cys Ser Ser 420 425 430 Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Lys Glu Ile Ser 435 440 445 Asn Thr Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn 450 455 460 Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu 465 470 475 480 Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys 485 490 495 Arg Ala Val Thr Leu Gly Ala Met Phe Leu Gly Phe Leu Gly Ala Ala 500 505 510 Gly Ser Thr Met Gly Ala Arg Ser Leu Thr Leu Thr Val Gln Ala Arg 515 520 525 Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala 530 535 540 Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys 545 550 555 560 Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln 565 570 575 Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr 580 585 590 Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Asp Gln Ile 595 600 605 Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr 610 615 620 Thr Asn Leu Ile Tyr Thr Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu 625 630 635 640 Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp 645 650 655 Asn Trp Phe Asp Ile Ser Lys Trp Leu Trp Tyr Ile Lys Ile Phe Ile 660 665 670 Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Thr Val Leu 675 680 685 Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln 690 695 700 Thr Arg Phe Pro Ala Pro Arg Gly Pro Asp Arg Pro Glu Gly Ile Glu 705 710 715 720 Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Ser Pro Leu Val His 725 730 735 Gly Leu Leu Ala Leu Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe 740 745 750 Ser Tyr His Arg Leu Arg Asp Leu Ile Leu Ile Ala Ala Arg Ile Val 755 760 765 Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Gly Asn 770 775 780 Leu Leu Gln Tyr Trp Ile Gln Glu Leu Lys Asn Ser Ala Val Ser Leu 785 790 795 800 Phe Asp Ala Ile Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Ile Ile 805 810 815 Glu Val Ala Gln Arg Ile Gly Arg Ala Phe Leu His Ile Pro Arg Arg

820 825 830 Ile Arg Gln Gly Phe Glu Arg Ala Leu Leu 835 840 3 867 PRT Artificial Sequence Description of Artificial Sequence TV1.8_2 3 Met Arg Val Met Gly Thr Gln Lys Asn Cys Gln Gln Trp Trp Ile Trp 1 5 10 15 Gly Ile Leu Gly Phe Trp Met Leu Met Ile Cys Asn Thr Glu Asp Leu 20 25 30 Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Asp Ala Lys Thr 35 40 45 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Thr Glu Val His 50 55 60 Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln 65 70 75 80 Glu Ile Val Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn 85 90 95 Asp Met Ala Asp Gln Met His Glu Asp Val Ile Ser Leu Trp Asp Gln 100 105 110 Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn 115 120 125 Cys Thr Asp Thr Asn Val Thr Gly Asn Arg Thr Val Thr Gly Asn Ser 130 135 140 Thr Asn Asn Thr Asn Gly Thr Gly Ile Tyr Asn Ile Glu Glu Met Lys 145 150 155 160 Asn Cys Ser Phe Asn Ala Thr Thr Glu Leu Arg Asp Lys Lys His Lys 165 170 175 Glu Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val Pro Leu Asn Glu Asn 180 185 190 Ser Asp Asn Phe Thr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Thr Ile 195 200 205 Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro Ile Pro Ile His Tyr 210 215 220 Cys Ala Pro Ala Gly Tyr Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe 225 230 235 240 Asn Gly Thr Gly Pro Cys Tyr Asn Val Ser Thr Val Gln Cys Thr His 245 250 255 Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu 260 265 270 Ala Glu Glu Gly Ile Ile Ile Arg Ser Glu Asn Leu Thr Glu Asn Thr 275 280 285 Lys Thr Ile Ile Val His Leu Asn Glu Ser Val Glu Ile Asn Cys Thr 290 295 300 Arg Pro Asn Asn Asn Thr Arg Lys Ser Val Arg Ile Gly Pro Gly Gln 305 310 315 320 Ala Phe Tyr Ala Thr Asn Asp Val Ile Gly Asn Ile Arg Gln Ala His 325 330 335 Cys Asn Ile Ser Thr Asp Arg Trp Asn Lys Thr Leu Gln Gln Val Met 340 345 350 Lys Lys Leu Gly Glu His Phe Pro Asn Lys Thr Ile Gln Phe Lys Pro 355 360 365 His Ala Gly Gly Asp Leu Glu Ile Thr Met His Ser Phe Asn Cys Arg 370 375 380 Gly Glu Phe Phe Tyr Cys Asn Thr Ser Asn Leu Phe Asn Ser Thr Tyr 385 390 395 400 His Ser Asn Asn Gly Thr Tyr Lys Tyr Asn Gly Asn Ser Ser Ser Pro 405 410 415 Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile Val Arg Met Trp Gln Gly 420 425 430 Val Gly Gln Ala Thr Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys 435 440 445 Arg Ser Asn Ile Thr Gly Ile Leu Leu Thr Arg Asp Gly Gly Phe Asn 450 455 460 Thr Thr Asn Asn Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg 465 470 475 480 Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys 485 490 495 Pro Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg 500 505 510 Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly 515 520 525 Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln 530 535 540 Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu 545 550 555 560 Lys Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly 565 570 575 Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys 580 585 590 Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Arg Leu Ile Cys 595 600 605 Thr Thr Ala Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Glu Lys 610 615 620 Asp Ile Trp Asp Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser 625 630 635 640 Asn Tyr Thr Gly Leu Ile Tyr Asn Leu Leu Glu Asp Ser Gln Asn Gln 645 650 655 Gln Glu Lys Asn Glu Lys Asp Leu Leu Glu Leu Asp Lys Trp Asn Asn 660 665 670 Leu Trp Asn Trp Phe Asp Ile Ser Asn Trp Pro Trp Tyr Ile Lys Ile 675 680 685 Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala 690 695 700 Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser 705 710 715 720 Phe Gln Thr Leu Thr Pro Ser Pro Arg Gly Leu Asp Arg Leu Gly Gly 725 730 735 Ile Glu Glu Glu Gly Gly Glu Gln Asp Arg Asp Arg Ser Ile Arg Leu 740 745 750 Val Ser Gly Phe Leu Ser Leu Ala Trp Asp Asp Leu Arg Asn Leu Cys 755 760 765 Leu Phe Ser Tyr His Arg Leu Arg Asp Phe Ile Leu Ile Ala Val Arg 770 775 780 Ala Val Glu Leu Leu Gly His Ser Ser Leu Arg Gly Leu Gln Arg Gly 785 790 795 800 Trp Glu Ile Leu Lys Tyr Leu Gly Ser Leu Val Gln Tyr Trp Gly Leu 805 810 815 Glu Leu Lys Lys Ser Ala Ile Ser Leu Leu Asp Thr Ile Ala Ile Thr 820 825 830 Val Ala Glu Gly Thr Asp Arg Ile Ile Glu Leu Val Gln Arg Ile Cys 835 840 845 Arg Ala Ile Leu Asn Ile Pro Arg Arg Ile Arg Gln Gly Phe Glu Ala 850 855 860 Ala Leu Leu 865 4 869 PRT Artificial Sequence Description of Artificial Sequence TV1.8_5 4 Met Arg Val Met Gly Thr Gln Lys Asn Cys Gln Gln Trp Trp Ile Trp 1 5 10 15 Gly Ile Leu Gly Phe Trp Met Leu Met Ile Cys Asn Thr Glu Asp Leu 20 25 30 Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala Lys Thr 35 40 45 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Thr Glu Val His 50 55 60 Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln 65 70 75 80 Glu Ile Val Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn 85 90 95 Asn Met Ala Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln 100 105 110 Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn 115 120 125 Cys Thr Asp Thr Asn Val Thr Gly Asn Arg Thr Val Thr Gly Asn Thr 130 135 140 Asn Asp Thr Asn Ile Ala Asn Ala Thr Tyr Lys Tyr Glu Glu Met Lys 145 150 155 160 Asn Cys Ser Phe Asn Ala Thr Thr Glu Leu Arg Asp Lys Lys His Lys 165 170 175 Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Ile Val Pro Leu Asn Glu Asn 180 185 190 Ser Asn Asn Phe Thr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Thr Ile 195 200 205 Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro Ile Pro Ile His Tyr 210 215 220 Cys Ala Pro Ala Asp Tyr Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe 225 230 235 240 Asn Gly Thr Gly Pro Cys Tyr Asn Val Ser Thr Val Gln Cys Thr His 245 250 255 Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu 260 265 270 Ala Glu Glu Gly Ile Ile Ile Arg Ser Glu Asn Leu Thr Glu Asn Thr 275 280 285 Lys Thr Ile Ile Val His Leu Asn Glu Ser Val Glu Ile Asn Cys Thr 290 295 300 Arg Pro Asn Asn Asn Thr Arg Lys Ser Val Arg Ile Gly Pro Gly Gln 305 310 315 320 Ala Phe Tyr Ala Thr Asn Asp Val Ile Gly Asn Ile Arg Gln Ala His 325 330 335 Cys Asn Ile Ser Thr Asp Arg Trp Asn Lys Thr Leu Gln Gln Val Met 340 345 350 Lys Lys Leu Gly Glu His Phe Pro Asn Lys Thr Ile Lys Phe Glu Pro 355 360 365 His Ala Gly Gly Asp Leu Glu Ile Thr Met His Ser Phe Asn Cys Arg 370 375 380 Gly Glu Phe Phe Tyr Cys Asn Thr Ser Asn Leu Phe Asn Ser Thr Tyr 385 390 395 400 Tyr Pro Lys Asn Gly Thr Tyr Lys Tyr Asn Gly Asn Ser Ser Leu Pro 405 410 415 Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile Val Arg Met Trp Gln Gly 420 425 430 Val Gly Gln Ala Met Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys 435 440 445 Arg Ser Asn Ile Thr Gly Ile Leu Leu Thr Arg Asp Gly Gly Phe Asn 450 455 460 Asn Thr Asn Asn Asp Thr Glu Glu Thr Phe Arg Pro Gly Gly Gly Asp 465 470 475 480 Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu 485 490 495 Ile Lys Pro Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg Arg Val Val 500 505 510 Gln Arg Lys Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe 515 520 525 Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr 530 535 540 Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn 545 550 555 560 Leu Leu Lys Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr Val 565 570 575 Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Ile Glu Arg Tyr 580 585 590 Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Arg Leu 595 600 605 Ile Cys Thr Thr Ala Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser 610 615 620 Glu Ala Asp Ile Trp Asp Asn Met Thr Trp Met Gln Trp Asp Arg Glu 625 630 635 640 Ile Asn Asn Tyr Thr Glu Thr Ile Phe Arg Leu Leu Glu Asp Ser Gln 645 650 655 Asn Gln Gln Glu Lys Asn Glu Lys Asp Leu Leu Glu Leu Asp Lys Trp 660 665 670 Asn Asn Leu Trp Asn Trp Phe Asp Ile Ser Asn Trp Leu Trp Tyr Ile 675 680 685 Lys Ile Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Ile 690 695 700 Phe Ala Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro 705 710 715 720 Leu Ser Phe Gln Thr Leu Thr Pro Ser Pro Arg Gly Leu Asp Arg Leu 725 730 735 Gly Gly Ile Glu Glu Glu Gly Gly Glu Gln Asp Arg Asp Arg Ser Ile 740 745 750 Arg Leu Val Ser Gly Phe Leu Ser Leu Ala Trp Asp Asp Leu Arg Ser 755 760 765 Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Phe Ile Leu Ile Ala 770 775 780 Val Arg Ala Val Glu Leu Leu Gly His Ser Ser Leu Arg Gly Leu Gln 785 790 795 800 Arg Gly Trp Glu Ile Leu Lys Tyr Leu Gly Ser Leu Val Gln Tyr Trp 805 810 815 Gly Leu Glu Leu Lys Lys Ser Ala Ile Ser Pro Leu Asp Thr Ile Ala 820 825 830 Ile Ala Val Ala Glu Gly Thr Asp Arg Ile Ile Glu Leu Val Gln Arg 835 840 845 Ile Cys Arg Ala Ile Leu Asn Ile Pro Arg Arg Ile Arg Gln Gly Phe 850 855 860 Glu Ala Ala Leu Leu 865 5 854 PRT Artificial Sequence Description of Artificial Sequence TV2.12-5/1 5 Met Arg Ala Arg Gly Ile Leu Lys Asn Tyr Arg His Trp Trp Ile Trp 1 5 10 15 Gly Ile Leu Gly Phe Trp Met Leu Met Met Cys Asn Val Lys Gly Leu 20 25 30 Trp Val Thr Val Tyr Tyr Gly Val Pro Val Gly Arg Glu Ala Lys Thr 35 40 45 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val His 50 55 60 Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln 65 70 75 80 Glu Val Ile Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn 85 90 95 Asp Met Val Asp Gln Met Gln Glu Asp Ile Ile Ser Leu Trp Asp Gln 100 105 110 Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn 115 120 125 Cys Thr Asn Ala Thr Val Asn Tyr Asn Asn Thr Ser Lys Asp Met Lys 130 135 140 Asn Cys Ser Phe Tyr Val Thr Thr Glu Leu Arg Asp Lys Lys Lys Lys 145 150 155 160 Glu Asn Ala Leu Phe Tyr Arg Leu Asp Ile Val Pro Leu Asn Asn Arg 165 170 175 Lys Asn Gly Asn Ile Asn Asn Tyr Arg Leu Ile Asn Cys Asn Thr Ser 180 185 190 Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro Ile Pro Ile 195 200 205 His Tyr Cys Ala Pro Ala Gly Tyr Ala Pro Leu Lys Cys Asn Asn Lys 210 215 220 Lys Phe Asn Gly Ile Gly Pro Cys Asp Asn Val Ser Thr Val Gln Cys 225 230 235 240 Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly 245 250 255 Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu Asn Leu Thr Asn 260 265 270 Asn Val Lys Thr Ile Ile Val His Leu Asn Glu Ser Ile Glu Ile Lys 275 280 285 Cys Thr Arg Pro Gly Asn Asn Thr Arg Lys Ser Val Arg Ile Gly Pro 290 295 300 Gly Gln Ala Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln 305 310 315 320 Ala His Cys Asn Ile Ser Lys Asn Glu Trp Asn Thr Thr Leu Gln Arg 325 330 335 Val Ser Gln Lys Leu Gln Glu Leu Phe Pro Asn Ser Thr Gly Ile Lys 340 345 350 Phe Ala Pro His Ser Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe 355 360 365 Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Asp Leu Phe Asn 370 375 380 Ser Thr Tyr Ser Asn Gly Thr Cys Thr Asn Gly Thr Cys Met Ser Asn 385 390 395 400 Asn Thr Glu Arg Ile Thr Leu Gln Cys Arg Ile Lys Gln Ile Ile Asn 405 410 415 Met Trp Gln Glu Val Gly Arg Ala Met Tyr Ala Pro Pro Ile Ala Gly 420 425 430 Asn Ile Thr Cys Arg Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp 435 440 445 Gly Gly Asp Asn Asn Thr Glu Thr Glu Thr Phe Arg Pro Gly Gly Gly 450 455 460 Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val 465 470 475 480 Glu Ile Lys Pro Leu Gly Val Ala Pro Thr Ala Ala Lys Arg Arg Val 485 490 495 Val Glu Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly 500 505 510 Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu 515 520 525 Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser 530 535 540 Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr 545 550 555 560 Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Ile Glu Arg 565 570 575 Tyr Leu Gln Asp Gln Gln Leu Leu Gly Leu Trp Gly Cys Ser Gly Lys 580 585 590 Leu Ile Cys Thr Thr Asn Val Leu Trp Asn Ser Ser Trp Ser Asn Lys 595 600 605 Thr Gln Ser Asp Ile Trp Asp Asn Met Thr Trp Met Gln Trp Asp Arg 610 615 620 Glu Ile Ser Asn Tyr Thr Asn Thr Ile Tyr Arg Leu Leu Glu Asp Ser 625 630 635 640 Gln Ser Gln Gln Glu Arg Asn Glu Lys Asp Leu Leu Ala Leu Asp Arg 645 650 655 Trp Asn Asn Leu Trp Asn Trp Phe Ser Ile Thr Asn Trp Leu Trp Tyr 660 665

670 Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile 675 680 685 Ile Phe Ala Val Leu Ser Leu Val Asn Arg Val Arg Gln Gly Tyr Ser 690 695 700 Pro Leu Ser Leu Gln Thr Leu Ile Pro Asn Pro Arg Gly Pro Asp Arg 705 710 715 720 Leu Gly Gly Ile Glu Glu Glu Gly Gly Glu Gln Asp Ser Ser Arg Ser 725 730 735 Ile Arg Leu Val Ser Gly Phe Leu Thr Leu Ala Trp Asp Asp Leu Arg 740 745 750 Ser Leu Cys Leu Phe Cys Tyr His Arg Leu Arg Asp Phe Ile Leu Ile 755 760 765 Val Val Arg Ala Val Glu Leu Leu Gly His Ser Ser Leu Arg Gly Leu 770 775 780 Gln Arg Gly Trp Gly Thr Leu Lys Tyr Leu Gly Ser Leu Val Gln Tyr 785 790 795 800 Trp Gly Leu Glu Leu Lys Lys Ser Ala Ile Asn Leu Leu Asp Thr Ile 805 810 815 Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Ile Leu Glu Phe Ile Gln 820 825 830 Asn Leu Cys Arg Gly Ile Arg Asn Val Pro Arg Arg Ile Arg Gln Gly 835 840 845 Phe Glu Ala Ala Leu Gln 850 6 860 PRT Artificial Sequence Description of Artificial Sequence consensus sequence 6 Met Arg Val Met Gly Thr Gln Lys Asn Cys Gln Gln Trp Trp Ile Trp 1 5 10 15 Gly Ile Leu Gly Phe Trp Met Leu Met Ile Cys Asn Val Glu Asp Leu 20 25 30 Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala Lys Thr 35 40 45 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Thr Glu Val His 50 55 60 Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln 65 70 75 80 Glu Ile Val Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn 85 90 95 Asn Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln 100 105 110 Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn 115 120 125 Cys Thr Asn Thr Asn Val Thr Gly Asn Arg Thr Val Thr Gly Asn Ser 130 135 140 Asn Ser Asn Ala Ala Tyr Glu Glu Met Lys Asn Cys Ser Phe Asn Val 145 150 155 160 Thr Thr Glu Leu Arg Asp Lys Lys His Lys Glu Tyr Ala Leu Phe Tyr 165 170 175 Lys Leu Asp Ile Val Pro Leu Asn Asn Glu Asn Ser Asn Asn Phe Thr 180 185 190 Tyr Arg Leu Ile Asn Cys Asn Thr Ser Thr Ile Thr Gln Ala Cys Pro 195 200 205 Lys Val Ser Phe Asp Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly 210 215 220 Tyr Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro 225 230 235 240 Cys Tyr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro Val 245 250 255 Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gly Ile 260 265 270 Ile Ile Arg Ser Glu Asn Leu Thr Glu Asn Thr Lys Thr Ile Ile Val 275 280 285 His Leu Asn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn 290 295 300 Thr Arg Lys Ser Val Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala Thr 305 310 315 320 Asn Asp Ile Ile Gly Asn Ile Arg Gln Ala His Cys Asn Ile Ser Thr 325 330 335 Asp Arg Trp Asn Lys Thr Leu Gln Gln Val Met Lys Lys Leu Gln Glu 340 345 350 His Phe Pro Asn Lys Thr Ile Lys Phe Lys Pro His Ala Gly Gly Asp 355 360 365 Leu Glu Ile Thr Met His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr 370 375 380 Cys Asn Thr Ser Asn Leu Phe Asn Ser Thr Tyr His Asn Asn Gly Thr 385 390 395 400 Tyr Lys Tyr Asn Gly Asn Ser Ser Pro Ile Thr Leu Gln Cys Lys Ile 405 410 415 Lys Gln Ile Ile Arg Met Trp Gln Gly Val Gly Gln Ala Met Tyr Ala 420 425 430 Pro Pro Ile Ala Gly Asn Ile Thr Cys Arg Ser Asn Ile Thr Gly Ile 435 440 445 Leu Leu Thr Arg Asp Gly Gly Phe Asn Asn Thr Asn Thr Thr Glu Thr 450 455 460 Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu 465 470 475 480 Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu Gly Ile Ala Pro Thr 485 490 495 Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala Val Gly Ile 500 505 510 Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 515 520 525 Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly 530 535 540 Ile Val Gln Gln Gln Ser Asn Leu Leu Lys Ala Ile Glu Ala Gln Gln 545 550 555 560 His Met Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg 565 570 575 Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile 580 585 590 Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn 595 600 605 Ser Ser Trp Ser Asn Lys Ser Glu Ala Asp Ile Trp Asp Asn Met Thr 610 615 620 Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr Thr Asn Thr Ile Tyr 625 630 635 640 Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu Lys Asn Glu Lys Asp 645 650 655 Leu Leu Glu Leu Asp Lys Trp Asn Asn Leu Trp Asn Trp Phe Asp Ile 660 665 670 Ser Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly 675 680 685 Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu Ser Ile Val Asn Arg 690 695 700 Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr Leu Thr Pro Ser 705 710 715 720 Pro Arg Gly Pro Asp Arg Leu Gly Gly Ile Glu Glu Glu Gly Gly Glu 725 730 735 Gln Asp Arg Asp Arg Ser Ile Arg Leu Val Ser Gly Phe Leu Ser Leu 740 745 750 Ala Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg Leu 755 760 765 Arg Asp Phe Ile Leu Ile Ala Val Arg Ala Val Glu Leu Leu Gly His 770 775 780 Ser Ser Leu Arg Gly Leu Gln Arg Gly Trp Glu Ile Leu Lys Tyr Leu 785 790 795 800 Gly Ser Leu Val Gln Tyr Trp Gly Leu Glu Leu Lys Lys Ser Ala Ile 805 810 815 Ser Leu Leu Asp Thr Ile Ala Ile Ala Val Ala Glu Gly Thr Asp Arg 820 825 830 Ile Ile Glu Leu Val Gln Arg Ile Cys Arg Ala Ile Leu Asn Ile Pro 835 840 845 Arg Arg Ile Arg Gln Gly Phe Glu Ala Ala Leu Leu 850 855 860 7 4 PRT Artificial Sequence Description of Artificial Sequence catalytic center 7 Tyr Met Asp Asp 1 8 4 PRT Artificial Sequence Description of Artificial Sequence primer grip region 8 Trp Met Gly Tyr 1 9 3999 DNA Artificial Sequence Description of Artificial Sequence GagComplPolmut.SF2 9 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgagggcca ccagatgaag gactgcaccg agcgccaggc caacttcctg 1320 ggcaagatct ggcccagcta caagggccgc cccggcaact tcctgcagag ccgccccgag 1380 cccaccgccc cccccgagga gagcttccgc ttcggcgagg agaagaccac ccccagccag 1440 aagcaggagc ccatcgacaa ggagctgtac cccctgacca gcctgcgcag cctgttcggc 1500 aacgacccca gcagccagaa agaattcaag gcccgcgtgc tggccgaggc gatgagccag 1560 gtgacgaacc cggcgaccat catgatgcag cgcggcaact tccgcaacca gcggaagacc 1620 gtcaagtgct tcaactgcgg caaggagggc cacaccgcca ggaactgccg cgccccccgc 1680 aagaagggct gctggcgctg cggccgcgaa ggacaccaaa tgaaagattg cactgagaga 1740 caggctaatt tcttccgcga ggacctggcc ttcctgcagg gcaaggcccg cgagttcagc 1800 agcgagcaga cccgcgccaa cagccccacc cgccgcgagc tgcaggtgtg gggcggcgag 1860 aacaacagcc tgagcgaggc cggcgccgac cgccagggca ccgtgagctt caacttcccc 1920 cagatcaccc tgtggcagcg ccccctggtg accatcagga tcggcggcca gctcaaggag 1980 gcgctgctcg acaccggcgc cgacgacacc gtgctggagg agatgaacct gcccggcaag 2040 tggaagccca agatgatcgg cgggatcggg ggcttcatca aggtgcggca gtacgaccag 2100 atccccgtgg agatctgcgg ccacaaggcc atcggcaccg tgctggtggg ccccaccccc 2160 gtgaacatca tcggccgcaa cctgctgacc cagatcggct gcaccctgaa cttccccatc 2220 agccccatcg agacggtgcc cgtgaagctg aagccgggga tggacggccc caaggtcaag 2280 cagtggcccc tgaccgagga gaagatcaag gccctggtgg agatctgcac cgagatggag 2340 aaggagggca agatcagcaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 2400 atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 2460 cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 2520 aagaagagcg tgaccgtgct ggacgtgggc gacgcctact tcagcgtgcc cctggacaag 2580 gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 2640 cgctaccagt acaacgtgct gccccagggc tggaagggca gccccgccat cttccagagc 2700 agcatgacca agatcctgga gcccttccgc aagcagaacc ccgacatcgt gatctaccag 2760 gcccccctgt acgtgggcag cgacctggag atcggccagc accgcaccaa gatcgaggag 2820 ctgcgccagc acctgctgcg ctggggcttc accacccccg acaagaagca ccagaaggag 2880 ccccccttcc tgcccatcga gctgcacccc gacaagtgga ccgtgcagcc catcatgctg 2940 cccgagaagg acagctggac cgtgaacgac atccagaagc tggtgggcaa gctgaactgg 3000 gccagccaga tctacgccgg catcaaggtg aagcagctgt gcaagctgct gcgcggcacc 3060 aaggccctga ccgaggtgat ccccctgacc gaggaggccg agctggagct ggccgagaac 3120 cgcgagatcc tgaaggagcc cgtgcacgag gtgtactacg accccagcaa ggacctggtg 3180 gccgagatcc agaagcaggg ccagggccag tggacctacc agatctacca ggagcccttc 3240 aagaacctga agaccggcaa gtacgcccgc atgcgcggcg cccacaccaa cgacgtgaag 3300 cagctgaccg aggccgtgca gaaggtgagc accgagagca tcgtgatctg gggcaagatc 3360 cccaagttca agctgcccat ccagaaggag acctgggagg cctggtggat ggagtactgg 3420 caggccacct ggatccccga gtgggagttc gtgaacaccc cccccctggt gaagctgtgg 3480 taccagctgg agaaggagcc catcgtgggc gccgagacct tctacgtgga cggcgccgcc 3540 aaccgcgaga ccaagctggg caaggccggc tacgtgaccg accggggccg gcagaaggtg 3600 gtgagcatcg ccgacaccac caaccagaag accgagctgc aggccatcca cctggccctg 3660 caggacagcg gcctggaggt gaacatcgtg accgacagcc agtacgccct gggcatcatc 3720 caggcccagc ccgacaagag cgagagcgag ctggtgagcc agatcatcga gcagctgatc 3780 aagaaggaga aggtgtacct ggcctgggtg cccgcccaca agggcatcgg cggcaacgag 3840 caggtggaca agctggtgag cgccggcatc cgcaaggtgc tgttcctgaa cggcatcgat 3900 ggcggcatcg tgatctacca gtacatggac gacctgtacg tgggcagcgg cggccctagg 3960 atcgattaaa agcttcccgg ggctagcacc ggttctaga 3999 10 3999 DNA Artificial Sequence Description of Artificial Sequence GagComplPolmutAtt.SF2 10 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgagggcca ccagatgaag gactgcaccg agcgccaggc caacttcctg 1320 ggcaagatct ggcccagcta caagggccgc cccggcaact tcctgcagag ccgccccgag 1380 cccaccgccc cccccgagga gagcttccgc ttcggcgagg agaagaccac ccccagccag 1440 aagcaggagc ccatcgacaa ggagctgtac cccctgacca gcctgcgcag cctgttcggc 1500 aacgacccca gcagccagaa agaattcaag gcccgcgtgc tggccgaggc gatgagccag 1560 gtgacgaacc cggcgaccat catgatgcag cgcggcaact tccgcaacca gcggaagacc 1620 gtcaagtgct tcaactgcgg caaggagggc cacaccgcca ggaactgccg cgccccccgc 1680 aagaagggct gctggcgctg cggccgcgaa ggacaccaaa tgaaagattg cactgagaga 1740 caggctaatt tcttccgcga ggacctggcc ttcctgcagg gcaaggcccg cgagttcagc 1800 agcgagcaga cccgcgccaa cagccccacc cgccgcgagc tgcaggtgtg gggcggcgag 1860 aacaacagcc tgagcgaggc cggcgccgac cgccagggca ccgtgagctt caacttcccc 1920 cagatcaccc tgtggcagcg ccccctggtg accatcagga tcggcggcca gctcaaggag 1980 gcgctgctcg actccggcgc cgacgacacc gtgctggagg agatgaacct gcccggcaag 2040 tggaagccca agatgatcgg cgggatcggg ggcttcatca aggtgcggca gtacgaccag 2100 atccccgtgg agatctgcgg ccacaaggcc atcggcaccg tgctggtggg ccccaccccc 2160 gtgaacatca tcggccgcaa cctgctgacc cagatcggct gcaccctgaa cttccccatc 2220 agccccatcg agacggtgcc cgtgaagctg aagccgggga tggacggccc caaggtcaag 2280 cagtggcccc tgaccgagga gaagatcaag gccctggtgg agatctgcac cgagatggag 2340 aaggagggca agatcagcaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 2400 atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 2460 cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 2520 aagaagagcg tgaccgtgct ggacgtgggc gacgcctact tcagcgtgcc cctggacaag 2580 gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 2640 cgctaccagt acaacgtgct gccccagggc tggaagggca gccccgccat cttccagagc 2700 agcatgacca agatcctgga gcccttccgc aagcagaacc ccgacatcgt gatctaccag 2760 gcccccctgt acgtgggcag cgacctggag atcggccagc accgcaccaa gatcgaggag 2820 ctgcgccagc acctgctgcg ctggggcttc accacccccg acaagaagca ccagaaggag 2880 ccccccttcc tgcccatcga gctgcacccc gacaagtgga ccgtgcagcc catcatgctg 2940 cccgagaagg acagctggac cgtgaacgac atccagaagc tggtgggcaa gctgaactgg 3000 gccagccaga tctacgccgg catcaaggtg aagcagctgt gcaagctgct gcgcggcacc 3060 aaggccctga ccgaggtgat ccccctgacc gaggaggccg agctggagct ggccgagaac 3120 cgcgagatcc tgaaggagcc cgtgcacgag gtgtactacg accccagcaa ggacctggtg 3180 gccgagatcc agaagcaggg ccagggccag tggacctacc agatctacca ggagcccttc 3240 aagaacctga agaccggcaa gtacgcccgc atgcgcggcg cccacaccaa cgacgtgaag 3300 cagctgaccg aggccgtgca gaaggtgagc accgagagca tcgtgatctg gggcaagatc 3360 cccaagttca agctgcccat ccagaaggag acctgggagg cctggtggat ggagtactgg 3420 caggccacct ggatccccga gtgggagttc gtgaacaccc cccccctggt gaagctgtgg 3480 taccagctgg agaaggagcc catcgtgggc gccgagacct tctacgtgga cggcgccgcc 3540 aaccgcgaga ccaagctggg caaggccggc tacgtgaccg accggggccg gcagaaggtg 3600 gtgagcatcg ccgacaccac caaccagaag accgagctgc aggccatcca cctggccctg 3660 caggacagcg gcctggaggt gaacatcgtg accgacagcc agtacgccct gggcatcatc 3720 caggcccagc ccgacaagag cgagagcgag ctggtgagcc agatcatcga gcagctgatc 3780 aagaaggaga aggtgtacct ggcctgggtg cccgcccaca agggcatcgg cggcaacgag 3840 caggtggaca agctggtgag cgccggcatc cgcaaggtgc tgttcctgaa cggcatcgat 3900 ggcggcatcg tgatctacca gtacatggac gacctgtacg tgggcagcgg cggccctagg 3960 atcgattaaa agcttcccgg ggctagcacc ggttctaga 3999 11 3999 DNA Artificial Sequence Description of Artificial Sequence GagComplPolmutIna.SF2 11 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca

agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgagggcca ccagatgaag gactgcaccg agcgccaggc caacttcctg 1320 ggcaagatct ggcccagcta caagggccgc cccggcaact tcctgcagag ccgccccgag 1380 cccaccgccc cccccgagga gagcttccgc ttcggcgagg agaagaccac ccccagccag 1440 aagcaggagc ccatcgacaa ggagctgtac cccctgacca gcctgcgcag cctgttcggc 1500 aacgacccca gcagccagaa agaattcaag gcccgcgtgc tggccgaggc gatgagccag 1560 gtgacgaacc cggcgaccat catgatgcag cgcggcaact tccgcaacca gcggaagacc 1620 gtcaagtgct tcaactgcgg caaggagggc cacaccgcca ggaactgccg cgccccccgc 1680 aagaagggct gctggcgctg cggccgcgaa ggacaccaaa tgaaagattg cactgagaga 1740 caggctaatt tcttccgcga ggacctggcc ttcctgcagg gcaaggcccg cgagttcagc 1800 agcgagcaga cccgcgccaa cagccccacc cgccgcgagc tgcaggtgtg gggcggcgag 1860 aacaacagcc tgagcgaggc cggcgccgac cgccagggca ccgtgagctt caacttcccc 1920 cagatcaccc tgtggcagcg ccccctggtg accatcagga tcggcggcca gctcaaggag 1980 gcgctgctcg ccaccggcgc cgacgacacc gtgctggagg agatgaacct gcccggcaag 2040 tggaagccca agatgatcgg cgggatcggg ggcttcatca aggtgcggca gtacgaccag 2100 atccccgtgg agatctgcgg ccacaaggcc atcggcaccg tgctggtggg ccccaccccc 2160 gtgaacatca tcggccgcaa cctgctgacc cagatcggct gcaccctgaa cttccccatc 2220 agccccatcg agacggtgcc cgtgaagctg aagccgggga tggacggccc caaggtcaag 2280 cagtggcccc tgaccgagga gaagatcaag gccctggtgg agatctgcac cgagatggag 2340 aaggagggca agatcagcaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 2400 atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 2460 cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 2520 aagaagagcg tgaccgtgct ggacgtgggc gacgcctact tcagcgtgcc cctggacaag 2580 gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 2640 cgctaccagt acaacgtgct gccccagggc tggaagggca gccccgccat cttccagagc 2700 agcatgacca agatcctgga gcccttccgc aagcagaacc ccgacatcgt gatctaccag 2760 gcccccctgt acgtgggcag cgacctggag atcggccagc accgcaccaa gatcgaggag 2820 ctgcgccagc acctgctgcg ctggggcttc accacccccg acaagaagca ccagaaggag 2880 ccccccttcc tgcccatcga gctgcacccc gacaagtgga ccgtgcagcc catcatgctg 2940 cccgagaagg acagctggac cgtgaacgac atccagaagc tggtgggcaa gctgaactgg 3000 gccagccaga tctacgccgg catcaaggtg aagcagctgt gcaagctgct gcgcggcacc 3060 aaggccctga ccgaggtgat ccccctgacc gaggaggccg agctggagct ggccgagaac 3120 cgcgagatcc tgaaggagcc cgtgcacgag gtgtactacg accccagcaa ggacctggtg 3180 gccgagatcc agaagcaggg ccagggccag tggacctacc agatctacca ggagcccttc 3240 aagaacctga agaccggcaa gtacgcccgc atgcgcggcg cccacaccaa cgacgtgaag 3300 cagctgaccg aggccgtgca gaaggtgagc accgagagca tcgtgatctg gggcaagatc 3360 cccaagttca agctgcccat ccagaaggag acctgggagg cctggtggat ggagtactgg 3420 caggccacct ggatccccga gtgggagttc gtgaacaccc cccccctggt gaagctgtgg 3480 taccagctgg agaaggagcc catcgtgggc gccgagacct tctacgtgga cggcgccgcc 3540 aaccgcgaga ccaagctggg caaggccggc tacgtgaccg accggggccg gcagaaggtg 3600 gtgagcatcg ccgacaccac caaccagaag accgagctgc aggccatcca cctggccctg 3660 caggacagcg gcctggaggt gaacatcgtg accgacagcc agtacgccct gggcatcatc 3720 caggcccagc ccgacaagag cgagagcgag ctggtgagcc agatcatcga gcagctgatc 3780 aagaaggaga aggtgtacct ggcctgggtg cccgcccaca agggcatcgg cggcaacgag 3840 caggtggaca agctggtgag cgccggcatc cgcaaggtgc tgttcctgaa cggcatcgat 3900 ggcggcatcg tgatctacca gtacatggac gacctgtacg tgggcagcgg cggccctagg 3960 atcgattaaa agcttcccgg ggctagcacc ggttctaga 3999 12 5274 DNA Artificial Sequence Description of Artificial Sequence gagCpolInaTatRevNef.opt_B 12 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgagggcca ccagatgaag gactgcaccg agcgccaggc caacttcctg 1320 ggcaagatct ggcccagcta caagggccgc cccggcaact tcctgcagag ccgccccgag 1380 cccaccgccc cccccgagga gagcttccgc ttcggcgagg agaagaccac ccccagccag 1440 aagcaggagc ccatcgacaa ggagctgtac cccctgacca gcctgcgcag cctgttcggc 1500 aacgacccca gcagccagaa agaattcaag gcccgcgtgc tggccgaggc gatgagccag 1560 gtgacgaacc cggcgaccat catgatgcag cgcggcaact tccgcaacca gcggaagacc 1620 gtcaagtgct tcaactgcgg caaggagggc cacaccgcca ggaactgccg cgccccccgc 1680 aagaagggct gctggcgctg cggccgcgaa ggacaccaaa tgaaagattg cactgagaga 1740 caggctaatt tcttccgcga ggacctggcc ttcctgcagg gcaaggcccg cgagttcagc 1800 agcgagcaga cccgcgccaa cagccccacc cgccgcgagc tgcaggtgtg gggcggcgag 1860 aacaacagcc tgagcgaggc cggcgccgac cgccagggca ccgtgagctt caacttcccc 1920 cagatcaccc tgtggcagcg ccccctggtg accatcagga tcggcggcca gctcaaggag 1980 gcgctgctcg ccaccggcgc cgacgacacc gtgctggagg agatgaacct gcccggcaag 2040 tggaagccca agatgatcgg cgggatcggg ggcttcatca aggtgcggca gtacgaccag 2100 atccccgtgg agatctgcgg ccacaaggcc atcggcaccg tgctggtggg ccccaccccc 2160 gtgaacatca tcggccgcaa cctgctgacc cagatcggct gcaccctgaa cttccccatc 2220 agccccatcg agacggtgcc cgtgaagctg aagccgggga tggacggccc caaggtcaag 2280 cagtggcccc tgaccgagga gaagatcaag gccctggtgg agatctgcac cgagatggag 2340 aaggagggca agatcagcaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 2400 atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 2460 cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 2520 aagaagagcg tgaccgtgct ggacgtgggc gacgcctact tcagcgtgcc cctggacaag 2580 gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 2640 cgctaccagt acaacgtgct gccccagggc tggaagggca gccccgccat cttccagagc 2700 agcatgacca agatcctgga gcccttccgc aagcagaacc ccgacatcgt gatctaccag 2760 gcccccctgt acgtgggcag cgacctggag atcggccagc accgcaccaa gatcgaggag 2820 ctgcgccagc acctgctgcg ctggggcttc accacccccg acaagaagca ccagaaggag 2880 ccccccttcc tgcccatcga gctgcacccc gacaagtgga ccgtgcagcc catcatgctg 2940 cccgagaagg acagctggac cgtgaacgac atccagaagc tggtgggcaa gctgaactgg 3000 gccagccaga tctacgccgg catcaaggtg aagcagctgt gcaagctgct gcgcggcacc 3060 aaggccctga ccgaggtgat ccccctgacc gaggaggccg agctggagct ggccgagaac 3120 cgcgagatcc tgaaggagcc cgtgcacgag gtgtactacg accccagcaa ggacctggtg 3180 gccgagatcc agaagcaggg ccagggccag tggacctacc agatctacca ggagcccttc 3240 aagaacctga agaccggcaa gtacgcccgc atgcgcggcg cccacaccaa cgacgtgaag 3300 cagctgaccg aggccgtgca gaaggtgagc accgagagca tcgtgatctg gggcaagatc 3360 cccaagttca agctgcccat ccagaaggag acctgggagg cctggtggat ggagtactgg 3420 caggccacct ggatccccga gtgggagttc gtgaacaccc cccccctggt gaagctgtgg 3480 taccagctgg agaaggagcc catcgtgggc gccgagacct tctacgtgga cggcgccgcc 3540 aaccgcgaga ccaagctggg caaggccggc tacgtgaccg accggggccg gcagaaggtg 3600 gtgagcatcg ccgacaccac caaccagaag accgagctgc aggccatcca cctggccctg 3660 caggacagcg gcctggaggt gaacatcgtg accgacagcc agtacgccct gggcatcatc 3720 caggcccagc ccgacaagag cgagagcgag ctggtgagcc agatcatcga gcagctgatc 3780 aagaaggaga aggtgtacct ggcctgggtg cccgcccaca agggcatcgg cggcaacgag 3840 caggtggaca agctggtgag cgccggcatc cgcaaggtgc tgttcctgaa cggcatcgat 3900 ggcggcatcg tgatctacca gtacatggac gacctgtacg tgggcagcgg cggccctagg 3960 gagcccgtgg acccccgcct ggagccctgg aagcaccccg gcagccagcc caagaccgcc 4020 ggcaccaact gctactgcaa gaagtgctgc ttccactgcc aggtgagctt catcaccaag 4080 ggcctgggca tcagctacgg ccgcaagaag cgccgccagc gccgccgcgc cccccccgac 4140 agcgaggtgc accaggtgag cctgcccaag cagcccgcca gccagcccca gggcgacccc 4200 accggcccca aggagagcaa gaagaaggtg gagcgcgaga ccgagaccga ccccgtgcac 4260 cccggggccg gccgcagcgg cgacagcgac gaggagctgc tgcagaccgt gcgcttcatc 4320 aagttcctgt accagagcaa ccccctgccc agccccaagg gcacccgcca ggccgacctg 4380 aaccgccgcc gccgctggcg cgagcgccag cgccagatcc agagcatcag cgcctggatc 4440 atcagcaccc acctgggccg cagcaccgag cccgtgcccc tgcagctgcc ccccgacctg 4500 cgcctgaacc tggactgcag cgaggactgc ggcaccagcg gcacccaggg cgtgggcagc 4560 ccccaggtgc tgggcgagag ccccgccgtg ctggacagcg gcaccaagga gctcgaggcc 4620 ggcaagtgga gcaagcgcat gagcggctgg agcgccgtgc gcgagcgcat gaagcgcgcc 4680 gagcccgccg agcccgccgc cgacggcgtg ggcgccgtga gccgcgacct ggagaagcac 4740 ggcgccatca ccagcagcaa caccgccgcc aacaacgccg actgcgcctg gctggaggcc 4800 caggaggacg aggacgtggg cttccccgtg cgcccccagg tgcccctgcg ccccatgacc 4860 tacaaggccg ccctggacct gagccacttc ctgaaggaga agggcggcct ggagggcctg 4920 atctacagcc agaagcgcca ggacatcctg gacctgtgga tccaccacac ccagggctac 4980 ttccccggct ggcagaacta cacccccggc cccggcatcc gctaccccct gaccttcggc 5040 tggtgcttca agctggtgcc cgtggacccc gactacgtgg aggaggccaa cgccggcgag 5100 aacaacagcc tgctgcaccc catgagccag cacggcatgg acgaccccga gaaggaggtg 5160 ctggtgtggc gcttcgacag ccgcctggcc ttccaccaca tggcccgcga gctgcacccc 5220 gagtactaca aggactgcga ttaaaagctt cccggggcta gcaccggttc taga 5274 13 3564 DNA Artificial Sequence Description of Artificial Sequence GagPolmutAtt.SF2 13 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgaaggaca ccaaatgaaa gattgcactg agagacaggc taatttcttc 1320 cgcgaggacc tggccttcct gcagggcaag gcccgcgagt tcagcagcga gcagacccgc 1380 gccaacagcc ccacccgccg cgagctgcag gtgtggggcg gcgagaacaa cagcctgagc 1440 gaggccggcg ccgaccgcca gggcaccgtg agcttcaact tcccccagat caccctgtgg 1500 cagcgccccc tggtgaccat caggatcggc ggccagctca aggaggcgct gctcgactcc 1560 ggcgccgacg acaccgtgct ggaggagatg aacctgcccg gcaagtggaa gcccaagatg 1620 atcggcggga tcgggggctt catcaaggtg cggcagtacg accagatccc cgtggagatc 1680 tgcggccaca aggccatcgg caccgtgctg gtgggcccca cccccgtgaa catcatcggc 1740 cgcaacctgc tgacccagat cggctgcacc ctgaacttcc ccatcagccc catcgagacg 1800 gtgcccgtga agctgaagcc ggggatggac ggccccaagg tcaagcagtg gcccctgacc 1860 gaggagaaga tcaaggccct ggtggagatc tgcaccgaga tggagaagga gggcaagatc 1920 agcaagatcg gccccgagaa cccctacaac acccccgtgt tcgccatcaa gaagaaggac 1980 agcaccaagt ggcgcaagct ggtggacttc cgcgagctga acaagcgcac ccaggacttc 2040 tgggaggtgc agctgggcat cccccacccc gccggcctga agaagaagaa gagcgtgacc 2100 gtgctggacg tgggcgacgc ctacttcagc gtgcccctgg acaaggactt ccgcaagtac 2160 accgccttca ccatccccag catcaacaac gagacccccg gcatccgcta ccagtacaac 2220 gtgctgcccc agggctggaa gggcagcccc gccatcttcc agagcagcat gaccaagatc 2280 ctggagccct tccgcaagca gaaccccgac atcgtgatct accaggcccc cctgtacgtg 2340 ggcagcgacc tggagatcgg ccagcaccgc accaagatcg aggagctgcg ccagcacctg 2400 ctgcgctggg gcttcaccac ccccgacaag aagcaccaga aggagccccc cttcctgccc 2460 atcgagctgc accccgacaa gtggaccgtg cagcccatca tgctgcccga gaaggacagc 2520 tggaccgtga acgacatcca gaagctggtg ggcaagctga actgggccag ccagatctac 2580 gccggcatca aggtgaagca gctgtgcaag ctgctgcgcg gcaccaaggc cctgaccgag 2640 gtgatccccc tgaccgagga ggccgagctg gagctggccg agaaccgcga gatcctgaag 2700 gagcccgtgc acgaggtgta ctacgacccc agcaaggacc tggtggccga gatccagaag 2760 cagggccagg gccagtggac ctaccagatc taccaggagc ccttcaagaa cctgaagacc 2820 ggcaagtacg cccgcatgcg cggcgcccac accaacgacg tgaagcagct gaccgaggcc 2880 gtgcagaagg tgagcaccga gagcatcgtg atctggggca agatccccaa gttcaagctg 2940 cccatccaga aggagacctg ggaggcctgg tggatggagt actggcaggc cacctggatc 3000 cccgagtggg agttcgtgaa cacccccccc ctggtgaagc tgtggtacca gctggagaag 3060 gagcccatcg tgggcgccga gaccttctac gtggacggcg ccgccaaccg cgagaccaag 3120 ctgggcaagg ccggctacgt gaccgaccgg ggccggcaga aggtggtgag catcgccgac 3180 accaccaacc agaagaccga gctgcaggcc atccacctgg ccctgcagga cagcggcctg 3240 gaggtgaaca tcgtgaccga cagccagtac gccctgggca tcatccaggc ccagcccgac 3300 aagagcgaga gcgagctggt gagccagatc atcgagcagc tgatcaagaa ggagaaggtg 3360 tacctggcct gggtgcccgc ccacaagggc atcggcggca acgagcaggt ggacaagctg 3420 gtgagcgccg gcatccgcaa ggtgctgttc ctgaacggca tcgatggcgg catcgtgatc 3480 taccagtaca tggacgacct gtacgtgggc agcggcggcc ctaggatcga ttaaaagctt 3540 cccggggcta gcaccggtga attc 3564 14 3564 DNA Artificial Sequence Description of Artificial Sequence GagPolmutIna.SF2 14 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgaaggaca ccaaatgaaa gattgcactg agagacaggc taatttcttc 1320 cgcgaggacc tggccttcct gcagggcaag gcccgcgagt tcagcagcga gcagacccgc 1380 gccaacagcc ccacccgccg cgagctgcag gtgtggggcg gcgagaacaa cagcctgagc 1440 gaggccggcg ccgaccgcca gggcaccgtg agcttcaact tcccccagat caccctgtgg 1500 cagcgccccc tggtgaccat caggatcggc ggccagctca aggaggcgct gctcgccacc 1560 ggcgccgacg acaccgtgct ggaggagatg aacctgcccg gcaagtggaa gcccaagatg 1620 atcggcggga tcgggggctt catcaaggtg cggcagtacg accagatccc cgtggagatc 1680 tgcggccaca aggccatcgg caccgtgctg gtgggcccca cccccgtgaa catcatcggc 1740 cgcaacctgc tgacccagat cggctgcacc ctgaacttcc ccatcagccc catcgagacg 1800 gtgcccgtga agctgaagcc ggggatggac ggccccaagg tcaagcagtg gcccctgacc 1860 gaggagaaga tcaaggccct ggtggagatc tgcaccgaga tggagaagga gggcaagatc 1920 agcaagatcg gccccgagaa cccctacaac acccccgtgt tcgccatcaa gaagaaggac 1980 agcaccaagt ggcgcaagct

ggtggacttc cgcgagctga acaagcgcac ccaggacttc 2040 tgggaggtgc agctgggcat cccccacccc gccggcctga agaagaagaa gagcgtgacc 2100 gtgctggacg tgggcgacgc ctacttcagc gtgcccctgg acaaggactt ccgcaagtac 2160 accgccttca ccatccccag catcaacaac gagacccccg gcatccgcta ccagtacaac 2220 gtgctgcccc agggctggaa gggcagcccc gccatcttcc agagcagcat gaccaagatc 2280 ctggagccct tccgcaagca gaaccccgac atcgtgatct accaggcccc cctgtacgtg 2340 ggcagcgacc tggagatcgg ccagcaccgc accaagatcg aggagctgcg ccagcacctg 2400 ctgcgctggg gcttcaccac ccccgacaag aagcaccaga aggagccccc cttcctgccc 2460 atcgagctgc accccgacaa gtggaccgtg cagcccatca tgctgcccga gaaggacagc 2520 tggaccgtga acgacatcca gaagctggtg ggcaagctga actgggccag ccagatctac 2580 gccggcatca aggtgaagca gctgtgcaag ctgctgcgcg gcaccaaggc cctgaccgag 2640 gtgatccccc tgaccgagga ggccgagctg gagctggccg agaaccgcga gatcctgaag 2700 gagcccgtgc acgaggtgta ctacgacccc agcaaggacc tggtggccga gatccagaag 2760 cagggccagg gccagtggac ctaccagatc taccaggagc ccttcaagaa cctgaagacc 2820 ggcaagtacg cccgcatgcg cggcgcccac accaacgacg tgaagcagct gaccgaggcc 2880 gtgcagaagg tgagcaccga gagcatcgtg atctggggca agatccccaa gttcaagctg 2940 cccatccaga aggagacctg ggaggcctgg tggatggagt actggcaggc cacctggatc 3000 cccgagtggg agttcgtgaa cacccccccc ctggtgaagc tgtggtacca gctggagaag 3060 gagcccatcg tgggcgccga gaccttctac gtggacggcg ccgccaaccg cgagaccaag 3120 ctgggcaagg ccggctacgt gaccgaccgg ggccggcaga aggtggtgag catcgccgac 3180 accaccaacc agaagaccga gctgcaggcc atccacctgg ccctgcagga cagcggcctg 3240 gaggtgaaca tcgtgaccga cagccagtac gccctgggca tcatccaggc ccagcccgac 3300 aagagcgaga gcgagctggt gagccagatc atcgagcagc tgatcaagaa ggagaaggtg 3360 tacctggcct gggtgcccgc ccacaagggc atcggcggca acgagcaggt ggacaagctg 3420 gtgagcgccg gcatccgcaa ggtgctgttc ctgaacggca tcgatggcgg catcgtgatc 3480 taccagtaca tggacgacct gtacgtgggc agcggcggcc ctaggatcga ttaaaagctt 3540 cccggggcta gcaccggtga attc 3564 15 3496 DNA Artificial Sequence Description of Artificial Sequence GagProtInaRTmut.SF2 15 gccaccatgg gcgcccgcgc cagcgtgctg agcggcggcg agctggacaa gtgggagaag 60 atccgcctgc gccccggcgg caagaagaag tacaagctga agcacatcgt gtgggccagc 120 cgcgagctgg agcgcttcgc cgtgaacccc ggcctgctgg agaccagcga gggctgccgc 180 cagatcctgg gccagctgca gcccagcctg cagaccggca gcgaggagct gcgcagcctg 240 tacaacaccg tggccaccct gtactgcgtg caccagcgca tcgacgtcaa ggacaccaag 300 gaggccctgg agaagatcga ggaggagcag aacaagtcca agaagaaggc ccagcaggcc 360 gccgccgccg ccggcaccgg caacagcagc caggtgagcc agaactaccc catcgtgcag 420 aacctgcagg gccagatggt gcaccaggcc atcagccccc gcaccctgaa cgcctgggtg 480 aaggtggtgg aggagaaggc cttcagcccc gaggtgatcc ccatgttcag cgccctgagc 540 gagggcgcca ccccccagga cctgaacacg atgttgaaca ccgtgggcgg ccaccaggcc 600 gccatgcaga tgctgaagga gaccatcaac gaggaggccg ccgagtggga ccgcgtgcac 660 cccgtgcacg ccggccccat cgcccccggc cagatgcgcg agccccgcgg cagcgacatc 720 gccggcacca ccagcaccct gcaggagcag atcggctgga tgaccaacaa cccccccatc 780 cccgtgggcg agatctacaa gcggtggatc atcctgggcc tgaacaagat cgtgcggatg 840 tacagcccca ccagcatcct ggacatccgc cagggcccca aggagccctt ccgcgactac 900 gtggaccgct tctacaagac cctgcgcgct gagcaggcca gccaggacgt gaagaactgg 960 atgaccgaga ccctgctggt gcagaacgcc aaccccgact gcaagaccat cctgaaggct 1020 ctcggccccg cggccaccct ggaggagatg atgaccgcct gccagggcgt gggcggcccc 1080 ggccacaagg cccgcgtgct ggccgaggcg atgagccagg tgacgaaccc ggcgaccatc 1140 atgatgcagc gcggcaactt ccgcaaccag cggaagaccg tcaagtgctt caactgcggc 1200 aaggagggcc acaccgccag gaactgccgc gccccccgca agaagggctg ctggcgctgc 1260 ggccgcgagg gccaccagat gaaggactgc accgagcgcc aggccaactt cctgggcaag 1320 atctggccca gctacaaggg ccgccccggc aacttcctgc agagccgccc cgagcccacc 1380 gccccccccg aggagagctt ccgcttcggc gaggagaaga ccacccccag ccagaagcag 1440 gagcccatcg acaaggagct gtaccccctg accagcctgc gcagcctgtt cggcaacgac 1500 cccagcagcc agaaagaatt cccccagatc accctgtggc agcgccccct ggtgaccatc 1560 aggatcggcg gccagctcaa ggaggcgctg ctcgccaccg gcgccgacga caccgtgctg 1620 gaggagatga acctgcccgg caagtggaag cccaagatga tcggcgggat cgggggcttc 1680 atcaaggtgc ggcagtacga ccagatcccc gtggagatct gcggccacaa ggccatcggc 1740 accgtgctgg tgggccccac ccccgtgaac atcatcggcc gcaacctgct gacccagatc 1800 ggctgcaccc tgaacttccc catcagcccc atcgagacgg tgcccgtgaa gctgaagccg 1860 gggatggacg gccccaaggt caagcagtgg cccctgaccg aggagaagat caaggccctg 1920 gtggagatct gcaccgagat ggagaaggag ggcaagatca gcaagatcgg ccccgagaac 1980 ccctacaaca cccccgtgtt cgccatcaag aagaaggaca gcaccaagtg gcgcaagctg 2040 gtggacttcc gcgagctgaa caagcgcacc caggacttct gggaggtgca gctgggcatc 2100 ccccaccccg ccggcctgaa gaagaagaag agcgtgaccg tgctggacgt gggcgacgcc 2160 tacttcagcg tgcccctgga caaggacttc cgcaagtaca ccgccttcac catccccagc 2220 atcaacaacg agacccccgg catccgctac cagtacaacg tgctgcccca gggctggaag 2280 ggcagccccg ccatcttcca gagcagcatg accaagatcc tggagccctt ccgcaagcag 2340 aaccccgaca tcgtgatcta ccaggccccc ctgtacgtgg gcagcgacct ggagatcggc 2400 cagcaccgca ccaagatcga ggagctgcgc cagcacctgc tgcgctgggg cttcaccacc 2460 cccgacaaga agcaccagaa ggagcccccc ttcctgccca tcgagctgca ccccgacaag 2520 tggaccgtgc agcccatcat gctgcccgag aaggacagct ggaccgtgaa cgacatccag 2580 aagctggtgg gcaagctgaa ctgggccagc cagatctacg ccggcatcaa ggtgaagcag 2640 ctgtgcaagc tgctgcgcgg caccaaggcc ctgaccgagg tgatccccct gaccgaggag 2700 gccgagctgg agctggccga gaaccgcgag atcctgaagg agcccgtgca cgaggtgtac 2760 tacgacccca gcaaggacct ggtggccgag atccagaagc agggccaggg ccagtggacc 2820 taccagatct accaggagcc cttcaagaac ctgaagaccg gcaagtacgc ccgcatgcgc 2880 ggcgcccaca ccaacgacgt gaagcagctg accgaggccg tgcagaaggt gagcaccgag 2940 agcatcgtga tctggggcaa gatccccaag ttcaagctgc ccatccagaa ggagacctgg 3000 gaggcctggt ggatggagta ctggcaggcc acctggatcc ccgagtggga gttcgtgaac 3060 accccccccc tggtgaagct gtggtaccag ctggagaagg agcccatcgt gggcgccgag 3120 accttctacg tggacggcgc cgccaaccgc gagaccaagc tgggcaaggc cggctacgtg 3180 accgaccggg gccggcagaa ggtggtgagc atcgccgaca ccaccaacca gaagaccgag 3240 ctgcaggcca tccacctggc cctgcaggac agcggcctgg aggtgaacat cgtgaccgac 3300 agccagtacg ccctgggcat catccaggcc cagcccgaca agagcgagag cgagctggtg 3360 agccagatca tcgagcagct gatcaagaag gagaaggtgt acctggcctg ggtgcccgcc 3420 cacaagggca tcggcggcaa cgagcaggtg gacaagctgg tgagcgccgg catccgcaag 3480 gtgctctaaa tctaga 3496 16 4773 DNA Artificial Sequence Description of Artificial Sequence GagProtInaRTmutTatRevNef.opt_B 16 gccaccatgg gcgcccgcgc cagcgtgctg agcggcggcg agctggacaa gtgggagaag 60 atccgcctgc gccccggcgg caagaagaag tacaagctga agcacatcgt gtgggccagc 120 cgcgagctgg agcgcttcgc cgtgaacccc ggcctgctgg agaccagcga gggctgccgc 180 cagatcctgg gccagctgca gcccagcctg cagaccggca gcgaggagct gcgcagcctg 240 tacaacaccg tggccaccct gtactgcgtg caccagcgca tcgacgtcaa ggacaccaag 300 gaggccctgg agaagatcga ggaggagcag aacaagtcca agaagaaggc ccagcaggcc 360 gccgccgccg ccggcaccgg caacagcagc caggtgagcc agaactaccc catcgtgcag 420 aacctgcagg gccagatggt gcaccaggcc atcagccccc gcaccctgaa cgcctgggtg 480 aaggtggtgg aggagaaggc cttcagcccc gaggtgatcc ccatgttcag cgccctgagc 540 gagggcgcca ccccccagga cctgaacacg atgttgaaca ccgtgggcgg ccaccaggcc 600 gccatgcaga tgctgaagga gaccatcaac gaggaggccg ccgagtggga ccgcgtgcac 660 cccgtgcacg ccggccccat cgcccccggc cagatgcgcg agccccgcgg cagcgacatc 720 gccggcacca ccagcaccct gcaggagcag atcggctgga tgaccaacaa cccccccatc 780 cccgtgggcg agatctacaa gcggtggatc atcctgggcc tgaacaagat cgtgcggatg 840 tacagcccca ccagcatcct ggacatccgc cagggcccca aggagccctt ccgcgactac 900 gtggaccgct tctacaagac cctgcgcgct gagcaggcca gccaggacgt gaagaactgg 960 atgaccgaga ccctgctggt gcagaacgcc aaccccgact gcaagaccat cctgaaggct 1020 ctcggccccg cggccaccct ggaggagatg atgaccgcct gccagggcgt gggcggcccc 1080 ggccacaagg cccgcgtgct ggccgaggcg atgagccagg tgacgaaccc ggcgaccatc 1140 atgatgcagc gcggcaactt ccgcaaccag cggaagaccg tcaagtgctt caactgcggc 1200 aaggagggcc acaccgccag gaactgccgc gccccccgca agaagggctg ctggcgctgc 1260 ggccgcgagg gccaccagat gaaggactgc accgagcgcc aggccaactt cctgggcaag 1320 atctggccca gctacaaggg ccgccccggc aacttcctgc agagccgccc cgagcccacc 1380 gccccccccg aggagagctt ccgcttcggc gaggagaaga ccacccccag ccagaagcag 1440 gagcccatcg acaaggagct gtaccccctg accagcctgc gcagcctgtt cggcaacgac 1500 cccagcagcc agaaagaatt cccccagatc accctgtggc agcgccccct ggtgaccatc 1560 aggatcggcg gccagctcaa ggaggcgctg ctcgccaccg gcgccgacga caccgtgctg 1620 gaggagatga acctgcccgg caagtggaag cccaagatga tcggcgggat cgggggcttc 1680 atcaaggtgc ggcagtacga ccagatcccc gtggagatct gcggccacaa ggccatcggc 1740 accgtgctgg tgggccccac ccccgtgaac atcatcggcc gcaacctgct gacccagatc 1800 ggctgcaccc tgaacttccc catcagcccc atcgagacgg tgcccgtgaa gctgaagccg 1860 gggatggacg gccccaaggt caagcagtgg cccctgaccg aggagaagat caaggccctg 1920 gtggagatct gcaccgagat ggagaaggag ggcaagatca gcaagatcgg ccccgagaac 1980 ccctacaaca cccccgtgtt cgccatcaag aagaaggaca gcaccaagtg gcgcaagctg 2040 gtggacttcc gcgagctgaa caagcgcacc caggacttct gggaggtgca gctgggcatc 2100 ccccaccccg ccggcctgaa gaagaagaag agcgtgaccg tgctggacgt gggcgacgcc 2160 tacttcagcg tgcccctgga caaggacttc cgcaagtaca ccgccttcac catccccagc 2220 atcaacaacg agacccccgg catccgctac cagtacaacg tgctgcccca gggctggaag 2280 ggcagccccg ccatcttcca gagcagcatg accaagatcc tggagccctt ccgcaagcag 2340 aaccccgaca tcgtgatcta ccaggccccc ctgtacgtgg gcagcgacct ggagatcggc 2400 cagcaccgca ccaagatcga ggagctgcgc cagcacctgc tgcgctgggg cttcaccacc 2460 cccgacaaga agcaccagaa ggagcccccc ttcctgccca tcgagctgca ccccgacaag 2520 tggaccgtgc agcccatcat gctgcccgag aaggacagct ggaccgtgaa cgacatccag 2580 aagctggtgg gcaagctgaa ctgggccagc cagatctacg ccggcatcaa ggtgaagcag 2640 ctgtgcaagc tgctgcgcgg caccaaggcc ctgaccgagg tgatccccct gaccgaggag 2700 gccgagctgg agctggccga gaaccgcgag atcctgaagg agcccgtgca cgaggtgtac 2760 tacgacccca gcaaggacct ggtggccgag atccagaagc agggccaggg ccagtggacc 2820 taccagatct accaggagcc cttcaagaac ctgaagaccg gcaagtacgc ccgcatgcgc 2880 ggcgcccaca ccaacgacgt gaagcagctg accgaggccg tgcagaaggt gagcaccgag 2940 agcatcgtga tctggggcaa gatccccaag ttcaagctgc ccatccagaa ggagacctgg 3000 gaggcctggt ggatggagta ctggcaggcc acctggatcc ccgagtggga gttcgtgaac 3060 accccccccc tggtgaagct gtggtaccag ctggagaagg agcccatcgt gggcgccgag 3120 accttctacg tggacggcgc cgccaaccgc gagaccaagc tgggcaaggc cggctacgtg 3180 accgaccggg gccggcagaa ggtggtgagc atcgccgaca ccaccaacca gaagaccgag 3240 ctgcaggcca tccacctggc cctgcaggac agcggcctgg aggtgaacat cgtgaccgac 3300 agccagtacg ccctgggcat catccaggcc cagcccgaca agagcgagag cgagctggtg 3360 agccagatca tcgagcagct gatcaagaag gagaaggtgt acctggcctg ggtgcccgcc 3420 cacaagggca tcggcggcaa cgagcaggtg gacaagctgg tgagcgccgg catccgcaag 3480 gtgctcaagc ttgagcccgt ggacccccgc ctggagccct ggaagcaccc cggcagccag 3540 cccaagaccg ccggcaccaa ctgctactgc aagaagtgct gcttccactg ccaggtgagc 3600 ttcatcacca agggcctggg catcagctac ggccgcaaga agcgccgcca gcgccgccgc 3660 gccccccccg acagcgaggt gcaccaggtg agcctgccca agcagcccgc cagccagccc 3720 cagggcgacc ccaccggccc caaggagagc aagaagaagg tggagcgcga gaccgagacc 3780 gaccccgtgc accccggggc cggccgcagc ggcgacagcg acgaggagct gctgcagacc 3840 gtgcgcttca tcaagttcct gtaccagagc aaccccctgc ccagccccaa gggcacccgc 3900 caggccgacc tgaaccgccg ccgccgctgg cgcgagcgcc agcgccagat ccagagcatc 3960 agcgcctgga tcatcagcac ccacctgggc cgcagcaccg agcccgtgcc cctgcagctg 4020 ccccccgacc tgcgcctgaa cctggactgc agcgaggact gcggcaccag cggcacccag 4080 ggcgtgggca gcccccaggt gctgggcgag agccccgccg tgctggacag cggcaccaag 4140 gagctcgagg ccggcaagtg gagcaagcgc atgagcggct ggagcgccgt gcgcgagcgc 4200 atgaagcgcg ccgagcccgc cgagcccgcc gccgacggcg tgggcgccgt gagccgcgac 4260 ctggagaagc acggcgccat caccagcagc aacaccgccg ccaacaacgc cgactgcgcc 4320 tggctggagg cccaggagga cgaggacgtg ggcttccccg tgcgccccca ggtgcccctg 4380 cgccccatga cctacaaggc cgccctggac ctgagccact tcctgaagga gaagggcggc 4440 ctggagggcc tgatctacag ccagaagcgc caggacatcc tggacctgtg gatccaccac 4500 acccagggct acttccccgg ctggcagaac tacacccccg gccccggcat ccgctacccc 4560 ctgaccttcg gctggtgctt caagctggtg cccgtggacc ccgactacgt ggaggaggcc 4620 aacgccggcg agaacaacag cctgctgcac cccatgagcc agcacggcat ggacgacccc 4680 gagaaggagg tgctggtgtg gcgcttcgac agccgcctgg ccttccacca catggcccgc 4740 gagctgcacc ccgagtacta caaggactgc taa 4773 17 3205 DNA Artificial Sequence Description of Artificial Sequence GagRTmut.SF2 17 gtcgacgcca ccatgggcgc ccgcgccagc gtgctgagcg gcggcgagct ggacaagtgg 60 gagaagatcc gcctgcgccc cggcggcaag aagaagtaca agctgaagca catcgtgtgg 120 gccagccgcg agctggagcg cttcgccgtg aaccccggcc tgctggagac cagcgagggc 180 tgccgccaga tcctgggcca gctgcagccc agcctgcaga ccggcagcga ggagctgcgc 240 agcctgtaca acaccgtggc caccctgtac tgcgtgcacc agcgcatcga cgtcaaggac 300 accaaggagg ccctggagaa gatcgaggag gagcagaaca agtccaagaa gaaggcccag 360 caggccgccg ccgccgccgg caccggcaac agcagccagg tgagccagaa ctaccccatc 420 gtgcagaacc tgcagggcca gatggtgcac caggccatca gcccccgcac cctgaacgcc 480 tgggtgaagg tggtggagga gaaggccttc agccccgagg tgatccccat gttcagcgcc 540 ctgagcgagg gcgccacccc ccaggacctg aacacgatgt tgaacaccgt gggcggccac 600 caggccgcca tgcagatgct gaaggagacc atcaacgagg aggccgccga gtgggaccgc 660 gtgcaccccg tgcacgccgg ccccatcgcc cccggccaga tgcgcgagcc ccgcggcagc 720 gacatcgccg gcaccaccag caccctgcag gagcagatcg gctggatgac caacaacccc 780 cccatccccg tgggcgagat ctacaagcgg tggatcatcc tgggcctgaa caagatcgtg 840 cggatgtaca gccccaccag catcctggac atccgccagg gccccaagga gcccttccgc 900 gactacgtgg accgcttcta caagaccctg cgcgctgagc aggccagcca ggacgtgaag 960 aactggatga ccgagaccct gctggtgcag aacgccaacc ccgactgcaa gaccatcctg 1020 aaggctctcg gccccgcggc caccctggag gagatgatga ccgcctgcca gggcgtgggc 1080 ggccccggcc acaaggcccg cgtgctggcc gaggcgatga gccaggtgac gaacccggcg 1140 accatcatga tgcagcgcgg caacttccgc aaccagcgga agaccgtcaa gtgcttcaac 1200 tgcggcaagg agggccacac cgccaggaac tgccgcgccc cccgcaagaa gggctgctgg 1260 cgctgcggcc gcgagggcca ccagatgaag gactgcaccg agcgccaggc caacttcctg 1320 ggcaagatct ggcccagcta caagggccgc cccggcaact tcctgcagag ccgccccgag 1380 cccaccgccc cccccgagga gagcttccgc ttcggcgagg agaagaccac ccccagccag 1440 aagcaggagc ccatcgacaa ggagctgtac cccctgacca gcctgcgcag cctgttcggc 1500 aacgacccca gcagccagaa agaattcccc atcagcccca tcgagacggt gcccgtgaag 1560 ctgaagccgg ggatggacgg ccccaaggtc aagcagtggc ccctgaccga ggagaagatc 1620 aaggccctgg tggagatctg caccgagatg gagaaggagg gcaagatcag caagatcggc 1680 cccgagaacc cctacaacac ccccgtgttc gccatcaaga agaaggacag caccaagtgg 1740 cgcaagctgg tggacttccg cgagctgaac aagcgcaccc aggacttctg ggaggtgcag 1800 ctgggcatcc cccaccccgc cggcctgaag aagaagaaga gcgtgaccgt gctggacgtg 1860 ggcgacgcct acttcagcgt gcccctggac aaggacttcc gcaagtacac cgccttcacc 1920 atccccagca tcaacaacga gacccccggc atccgctacc agtacaacgt gctgccccag 1980 ggctggaagg gcagccccgc catcttccag agcagcatga ccaagatcct ggagcccttc 2040 cgcaagcaga accccgacat cgtgatctac caggcccccc tgtacgtggg cagcgacctg 2100 gagatcggcc agcaccgcac caagatcgag gagctgcgcc agcacctgct gcgctggggc 2160 ttcaccaccc ccgacaagaa gcaccagaag gagcccccct tcctgcccat cgagctgcac 2220 cccgacaagt ggaccgtgca gcccatcatg ctgcccgaga aggacagctg gaccgtgaac 2280 gacatccaga agctggtggg caagctgaac tgggccagcc agatctacgc cggcatcaag 2340 gtgaagcagc tgtgcaagct gctgcgcggc accaaggccc tgaccgaggt gatccccctg 2400 accgaggagg ccgagctgga gctggccgag aaccgcgaga tcctgaagga gcccgtgcac 2460 gaggtgtact acgaccccag caaggacctg gtggccgaga tccagaagca gggccagggc 2520 cagtggacct accagatcta ccaggagccc ttcaagaacc tgaagaccgg caagtacgcc 2580 cgcatgcgcg gcgcccacac caacgacgtg aagcagctga ccgaggccgt gcagaaggtg 2640 agcaccgaga gcatcgtgat ctggggcaag atccccaagt tcaagctgcc catccagaag 2700 gagacctggg aggcctggtg gatggagtac tggcaggcca cctggatccc cgagtgggag 2760 ttcgtgaaca ccccccccct ggtgaagctg tggtaccagc tggagaagga gcccatcgtg 2820 ggcgccgaga ccttctacgt ggacggcgcc gccaaccgcg agaccaagct gggcaaggcc 2880 ggctacgtga ccgaccgggg ccggcagaag gtggtgagca tcgccgacac caccaaccag 2940 aagaccgagc tgcaggccat ccacctggcc ctgcaggaca gcggcctgga ggtgaacatc 3000 gtgaccgaca gccagtacgc cctgggcatc atccaggccc agcccgacaa gagcgagagc 3060 gagctggtga gccagatcat cgagcagctg atcaagaagg agaaggtgta cctggcctgg 3120 gtgcccgccc acaagggcat cggcggcaac gagcaggtgg acaagctggt gagcgccggc 3180 atccgcaagg tgctctaaat ctaga 3205 18 2799 DNA Artificial Sequence Description of Artificial Sequence GagTatRevNef.opt_B 18 gccaccatgg gcgcccgcgc cagcgtgctg agcggcggcg agctggacaa gtgggagaag 60 atccgcctgc gccccggcgg caagaagaag tacaagctga agcacatcgt gtgggccagc 120 cgcgagctgg agcgcttcgc cgtgaacccc ggcctgctgg agaccagcga gggctgccgc 180 cagatcctgg gccagctgca gcccagcctg cagaccggca gcgaggagct gcgcagcctg 240 tacaacaccg tggccaccct gtactgcgtg caccagcgca tcgacgtcaa ggacaccaag 300 gaggccctgg agaagatcga ggaggagcag aacaagtcca agaagaaggc ccagcaggcc 360 gccgccgccg ccggcaccgg caacagcagc caggtgagcc agaactaccc catcgtgcag 420 aacctgcagg gccagatggt gcaccaggcc atcagccccc gcaccctgaa cgcctgggtg 480 aaggtggtgg aggagaaggc cttcagcccc gaggtgatcc ccatgttcag cgccctgagc 540 gagggcgcca ccccccagga cctgaacacg atgttgaaca ccgtgggcgg ccaccaggcc 600 gccatgcaga tgctgaagga gaccatcaac gaggaggccg ccgagtggga ccgcgtgcac 660 cccgtgcacg ccggccccat cgcccccggc cagatgcgcg agccccgcgg cagcgacatc 720 gccggcacca ccagcaccct gcaggagcag atcggctgga tgaccaacaa cccccccatc 780 cccgtgggcg agatctacaa gcggtggatc atcctgggcc tgaacaagat cgtgcggatg 840 tacagcccca ccagcatcct ggacatccgc cagggcccca aggagccctt ccgcgactac 900 gtggaccgct tctacaagac cctgcgcgct gagcaggcca gccaggacgt gaagaactgg 960 atgaccgaga ccctgctggt gcagaacgcc aaccccgact gcaagaccat cctgaaggct 1020 ctcggccccg cggccaccct ggaggagatg atgaccgcct gccagggcgt gggcggcccc 1080 ggccacaagg cccgcgtgct ggccgaggcg atgagccagg tgacgaaccc ggcgaccatc 1140 atgatgcagc gcggcaactt ccgcaaccag cggaagaccg tcaagtgctt caactgcggc 1200 aaggagggcc acaccgccag gaactgccgc gccccccgca agaagggctg ctggcgctgc 1260 ggccgcgagg gccaccagat gaaggactgc accgagcgcc aggccaactt cctgggcaag 1320 atctggccca gctacaaggg ccgccccggc aacttcctgc agagccgccc cgagcccacc 1380 gccccccccg aggagagctt ccgcttcggc gaggagaaga ccacccccag ccagaagcag 1440 gagcccatcg acaaggagct gtaccccctg accagcctgc gcagcctgtt cggcaacgac 1500 cccagcagcc aggaattcga gcccgtggac ccccgcctgg

agccctggaa gcaccccggc 1560 agccagccca agaccgccgg caccaactgc tactgcaaga agtgctgctt ccactgccag 1620 gtgagcttca tcaccaaggg cctgggcatc agctacggcc gcaagaagcg ccgccagcgc 1680 cgccgcgccc cccccgacag cgaggtgcac caggtgagcc tgcccaagca gcccgccagc 1740 cagccccagg gcgaccccac cggccccaag gagagcaaga agaaggtgga gcgcgagacc 1800 gagaccgacc ccgtgcaccc cggggccggc cgcagcggcg acagcgacga ggagctgctg 1860 cagaccgtgc gcttcatcaa gttcctgtac cagagcaacc ccctgcccag ccccaagggc 1920 acccgccagg ccgacctgaa ccgccgccgc cgctggcgcg agcgccagcg ccagatccag 1980 agcatcagcg cctggatcat cagcacccac ctgggccgca gcaccgagcc cgtgcccctg 2040 cagctgcccc ccgacctgcg cctgaacctg gactgcagcg aggactgcgg caccagcggc 2100 acccagggcg tgggcagccc ccaggtgctg ggcgagagcc ccgccgtgct ggacagcggc 2160 accaaggagc tcgaggccgg caagtggagc aagcgcatga gcggctggag cgccgtgcgc 2220 gagcgcatga agcgcgccga gcccgccgag cccgccgccg acggcgtggg cgccgtgagc 2280 cgcgacctgg agaagcacgg cgccatcacc agcagcaaca ccgccgccaa caacgccgac 2340 tgcgcctggc tggaggccca ggaggacgag gacgtgggct tccccgtgcg cccccaggtg 2400 cccctgcgcc ccatgaccta caaggccgcc ctggacctga gccacttcct gaaggagaag 2460 ggcggcctgg agggcctgat ctacagccag aagcgccagg acatcctgga cctgtggatc 2520 caccacaccc agggctactt ccccggctgg cagaactaca cccccggccc cggcatccgc 2580 taccccctga ccttcggctg gtgcttcaag ctggtgcccg tggaccccga ctacgtggag 2640 gaggccaacg ccggcgagaa caacagcctg ctgcacccca tgagccagca cggcatggac 2700 gaccccgaga aggaggtgct ggtgtggcgc ttcgacagcc gcctggcctt ccaccacatg 2760 gcccgcgagc tgcaccccga gtactacaag gactgctaa 2799 19 2028 DNA Artificial Sequence Description of Artificial Sequence gp140.modSF162.CwtLmod 19 atgcgcgtga tgggcaccca gaagaactgc cagcagtggt ggatctgggg catcctgggc 60 ttctggatgc tgatgatctg cagcgccgtg gagaagctgt gggtgaccgt gtactacggc 120 gtgcccgtgt ggaaggaggc caccaccacc ctgttctgcg ccagcgacgc caaggcctac 180 gacaccgagg tgcacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 240 caggagatcg tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa caacatggtg 300 gagcagatgc acgaggacat catcagcctg tgggaccaga gcctgaagcc ctgcgtgaag 360 ctgacccccc tgtgcgtgac cctgcactgc accaacctga agaacgccac caacaccaag 420 agcagcaact ggaaggagat ggaccgcggc gagatcaaga actgcagctt caaggtgacc 480 accagcatcc gcaacaagat gcagaaggag tacgccctgt tctacaagct ggacgtggtg 540 cccatcgaca acgacaacac cagctacaag ctgatcaact gcaacaccag cgtgatcacc 600 caggcctgcc ccaaggtgag cttcgagccc atccccatcc actactgcgc ccccgccggc 660 ttcgccatcc tgaagtgcaa cgacaagaag ttcaacggca gcggcccctg caccaacgtg 720 agcaccgtgc agtgcaccca cggcatccgc cccgtggtga gcacccagct gctgctgaac 780 ggcagcctgg ccgaggaggg cgtggtgatc cgcagcgaga acttcaccga caacgccaag 840 accatcatcg tgcagctgaa ggagagcgtg gagatcaact gcacccgccc caacaacaac 900 acccgcaaga gcatcaccat cggccccggc cgcgccttct acgccaccgg cgacatcatc 960 ggcgacatcc gccaggccca ctgcaacatc agcggcgaga agtggaacaa caccctgaag 1020 cagatcgtga ccaagctgca ggcccagttc ggcaacaaga ccatcgtgtt caagcagagc 1080 agcggcggcg accccgagat cgtgatgcac agcttcaact gcggcggcga gttcttctac 1140 tgcaacagca cccagctgtt caacagcacc tggaacaaca ccatcggccc caacaacacc 1200 aacggcacca tcaccctgcc ctgccgcatc aagcagatca tcaaccgctg gcaggaggtg 1260 ggcaaggcca tgtacgcccc ccccatccgc ggccagatcc gctgcagcag caacatcacc 1320 ggcctgctgc tgacccgcga cggcggcaag gagatcagca acaccaccga gatcttccgc 1380 cccggcggcg gcgacatgcg cgacaactgg cgcagcgagc tgtacaagta caaggtggtg 1440 aagatcgagc ccctgggcgt ggcccccacc aaggccaagc gccgcgtggt gcagcgcgag 1500 aagcgcgccg tgaccctggg cgccatgttc ctgggcttcc tgggcgccgc cggcagcacc 1560 atgggcgccc gcagcctgac cctgaccgtg caggcccgcc agctgctgag cggcatcgtg 1620 cagcagcaga acaacctgct gcgcgccatc gaggcccagc agcacctgct gcagctgacc 1680 gtgtggggca tcaagcagct gcaggcccgc gtgctggccg tggagcgcta cctgaaggac 1740 cagcagctgc tgggcatctg gggctgcagc ggcaagctga tctgcaccac cgccgtgccc 1800 tggaacgcca gctggagcaa caagagcctg gaccagatct ggaacaacat gacctggatg 1860 gagtgggagc gcgagatcga caactacacc aacctgatct acaccctgat cgaggagagc 1920 cagaaccagc aggagaagaa cgagcaggag ctgctggagc tggacaagtg ggccagcctg 1980 tggaactggt tcgacatcag caagtggctg tggtacatct aactcgag 2028 20 2033 DNA Artificial Sequence Description of Artificial Sequence gp140.modSF162.CwtLnat 20 atgagagtga tggggacaca gaagaattgt caacaatggt ggatatgggg catcttaggc 60 ttctggatgc taatgatttg tagcgccgtg gagaagctgt gggtgaccgt gtactacggc 120 gtgcccgtgt ggaaggaggc caccaccacc ctgttctgcg ccagcgacgc caaggcctac 180 gacaccgagg tgcacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 240 caggagatcg tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa caacatggtg 300 gagcagatgc acgaggacat catcagcctg tgggaccaga gcctgaagcc ctgcgtgaag 360 ctgacccccc tgtgcgtgac cctgcactgc accaacctga agaacgccac caacaccaag 420 agcagcaact ggaaggagat ggaccgcggc gagatcaaga actgcagctt caaggtgacc 480 accagcatcc gcaacaagat gcagaaggag tacgccctgt tctacaagct ggacgtggtg 540 cccatcgaca acgacaacac cagctacaag ctgatcaact gcaacaccag cgtgatcacc 600 caggcctgcc ccaaggtgag cttcgagccc atccccatcc actactgcgc ccccgccggc 660 ttcgccatcc tgaagtgcaa cgacaagaag ttcaacggca gcggcccctg caccaacgtg 720 agcaccgtgc agtgcaccca cggcatccgc cccgtggtga gcacccagct gctgctgaac 780 ggcagcctgg ccgaggaggg cgtggtgatc cgcagcgaga acttcaccga caacgccaag 840 accatcatcg tgcagctgaa ggagagcgtg gagatcaact gcacccgccc caacaacaac 900 acccgcaaga gcatcaccat cggccccggc cgcgccttct acgccaccgg cgacatcatc 960 ggcgacatcc gccaggccca ctgcaacatc agcggcgaga agtggaacaa caccctgaag 1020 cagatcgtga ccaagctgca ggcccagttc ggcaacaaga ccatcgtgtt caagcagagc 1080 agcggcggcg accccgagat cgtgatgcac agcttcaact gcggcggcga gttcttctac 1140 tgcaacagca cccagctgtt caacagcacc tggaacaaca ccatcggccc caacaacacc 1200 aacggcacca tcaccctgcc ctgccgcatc aagcagatca tcaaccgctg gcaggaggtg 1260 ggcaaggcca tgtacgcccc ccccatccgc ggccagatcc gctgcagcag caacatcacc 1320 ggcctgctgc tgacccgcga cggcggcaag gagatcagca acaccaccga gatcttccgc 1380 cccggcggcg gcgacatgcg cgacaactgg cgcagcgagc tgtacaagta caaggtggtg 1440 aagatcgagc ccctgggcgt ggcccccacc aaggccaagc gccgcgtggt gcagcgcgag 1500 aagcgcgccg tgaccctggg cgccatgttc ctgggcttcc tgggcgccgc cggcagcacc 1560 atgggcgccc gcagcctgac cctgaccgtg caggcccgcc agctgctgag cggcatcgtg 1620 cagcagcaga acaacctgct gcgcgccatc gaggcccagc agcacctgct gcagctgacc 1680 gtgtggggca tcaagcagct gcaggcccgc gtgctggccg tggagcgcta cctgaaggac 1740 cagcagctgc tgggcatctg gggctgcagc ggcaagctga tctgcaccac cgccgtgccc 1800 tggaacgcca gctggagcaa caagagcctg gaccagatct ggaacaacat gacctggatg 1860 gagtgggagc gcgagatcga caactacacc aacctgatct acaccctgat cgaggagagc 1920 cagaaccagc aggagaagaa cgagcaggag ctgctggagc tggacaagtg ggccagcctg 1980 tggaactggt tcgacatcag caagtggctg tggtacatct aactcgaggr sht 2033 21 2453 DNA Artificial Sequence Description of Artificial Sequence gp160.modSF162.delV2.mut7 21 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccagcg ccgtggagaa gctgtgggtg accgtgtact acggcgtgcc cgtgtggaag 120 gaggccacca ccaccctgtt ctgcgccagc gacgccaagg cctacgacac cgaggtgcac 180 aacgtgtggg ccacccacgc ctgcgtgccc accgacccca acccccagga gatcgtgctg 240 gagaacgtga ccgagaactt caacatgtgg aagaacaaca tggtggagca gatgcacgag 300 gacatcatca gcctgtggga ccagagcctg aagccctgcg tgaagctgac ccccctgtgc 360 gtgaccctgc actgcaccaa cctgaagaac gccaccaaca ccaagagcag caactggaag 420 gagatggacc gcggcgagat caagaactgc agcttcaagg tgggcgccgg caagctgatc 480 aactgcaaca ccagcgtgat cacccaggcc tgccccaagg tgagcttcga gcccatcccc 540 atccactact gcgcccccgc cggcttcgcc atcctgaagt gcaacgacaa gaagttcaac 600 ggcagcggcc cctgcaccaa cgtgagcacc gtgcagtgca cccacggcat ccgccccgtg 660 gtgagcaccc agctgctgct gaacggcagc ctggccgagg agggcgtggt gatccgcagc 720 gagaacttca ccgacaacgc caagaccatc atcgtgcagc tgaaggagag cgtggagatc 780 aactgcaccc gccccaacaa caacacccgc aagagcatca ccatcggccc cggccgcgcc 840 ttctacgcca ccggcgacat catcggcgac atccgccagg cccactgcaa catcagcggc 900 gagaagtgga acaacaccct gaagcagatc gtgaccaagc tgcaggccca gttcggcaac 960 aagaccatcg tgttcaagca gagcagcggc ggcgaccccg agatcgtgat gcacagcttc 1020 aactgcggcg gcgagttctt ctactgcaac agcacccagc tgttcaacag cacctggaac 1080 aacaccatcg gccccaacaa caccaacggc accatcaccc tgccctgccg catcaagcag 1140 atcatcaacc gctggcagga ggtgggcaag gccatgtacg ccccccccat ccgcggccag 1200 atccgctgca gcagcaacat caccggcctg ctgctgaccc gcgacggcgg caaggagatc 1260 agcaacacca ccgagatctt ccgccccggc ggcggcgaca tgcgcgacaa ctggcgcagc 1320 gagctgtaca agtacaaggt ggtgaagatc gagcccctgg gcgtggcccc caccaaggcc 1380 atcagcagcg tggtgcagag cgagaagagc gccgtgaccc tgggcgccat gttcctgggc 1440 ttcctgggcg ccgccggcag caccatgggc gcccgcagcc tgaccctgac cgtgcaggcc 1500 cgccagctgc tgagcggcat cgtgcagcag cagaacaacc tgctgcgcgc catcgaggcc 1560 cagcagcacc tgctgcagct gaccgtgtgg ggcatcaagc agctgcaggc ccgcgtgctg 1620 gccgtggagc gctacctgaa ggaccagcag ctgctgggca tctggggctg cagcggcaag 1680 ctgatctgca ccaccgccgt gccctggaac gccagctgga gcaacaagag cctggaccag 1740 atctggaaca acatgacctg gatggagtgg gagcgcgaga tcgacaacta caccaacctg 1800 atctacaccc tgatcgagga gagccagaac cagcaggaga agaacgagca ggagctgctg 1860 gagctggaca agtgggccag cctgtggaac tggttcgaca tcagcaagtg gctgtggtac 1920 atcaagatct tcatcatgat cgtgggcggc ctggtgggcc tgcgcatcgt gttcaccgtg 1980 ctgagcatcg tgaaccgcgt gcgccagggc tacagccccc tgagcttcca gacccgcttc 2040 cccgcccccc gcggccccga ccgccccgag ggcatcgagg aggagggcgg cgagcgcgac 2100 cgcgaccgca gcagccccct ggtgcacggc ctgctggccc tgatctggga cgacctgcgc 2160 agcctgtgcc tgttcagcta ccaccgcctg cgcgacctga tcctgatcgc cgcccgcatc 2220 gtggagctgc tgggccgccg cggctgggag gccctgaagt actggggcaa cctgctgcag 2280 tactggatcc aggagctgaa gaacagcgcc gtgagcctgt tcgacgccat cgccatcgcc 2340 gtggccgagg gcaccgaccg catcatcgag gtggcccagc gcatcggccg cgccttcctg 2400 cacatccccc gccgcatccg ccagggcttc gagcgcgccc tgctgtaagr sht 2453 22 2453 DNA Artificial Sequence Description of Artificial Sequence gp160.modSF162.delV2.mut8 22 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccagcg ccgtggagaa gctgtgggtg accgtgtact acggcgtgcc cgtgtggaag 120 gaggccacca ccaccctgtt ctgcgccagc gacgccaagg cctacgacac cgaggtgcac 180 aacgtgtggg ccacccacgc ctgcgtgccc accgacccca acccccagga gatcgtgctg 240 gagaacgtga ccgagaactt caacatgtgg aagaacaaca tggtggagca gatgcacgag 300 gacatcatca gcctgtggga ccagagcctg aagccctgcg tgaagctgac ccccctgtgc 360 gtgaccctgc actgcaccaa cctgaagaac gccaccaaca ccaagagcag caactggaag 420 gagatggacc gcggcgagat caagaactgc agcttcaagg tgggcgccgg caagctgatc 480 aactgcaaca ccagcgtgat cacccaggcc tgccccaagg tgagcttcga gcccatcccc 540 atccactact gcgcccccgc cggcttcgcc atcctgaagt gcaacgacaa gaagttcaac 600 ggcagcggcc cctgcaccaa cgtgagcacc gtgcagtgca cccacggcat ccgccccgtg 660 gtgagcaccc agctgctgct gaacggcagc ctggccgagg agggcgtggt gatccgcagc 720 gagaacttca ccgacaacgc caagaccatc atcgtgcagc tgaaggagag cgtggagatc 780 aactgcaccc gccccaacaa caacacccgc aagagcatca ccatcggccc cggccgcgcc 840 ttctacgcca ccggcgacat catcggcgac atccgccagg cccactgcaa catcagcggc 900 gagaagtgga acaacaccct gaagcagatc gtgaccaagc tgcaggccca gttcggcaac 960 aagaccatcg tgttcaagca gagcagcggc ggcgaccccg agatcgtgat gcacagcttc 1020 aactgcggcg gcgagttctt ctactgcaac agcacccagc tgttcaacag cacctggaac 1080 aacaccatcg gccccaacaa caccaacggc accatcaccc tgccctgccg catcaagcag 1140 atcatcaacc gctggcagga ggtgggcaag gccatgtacg ccccccccat ccgcggccag 1200 atccgctgca gcagcaacat caccggcctg ctgctgaccc gcgacggcgg caaggagatc 1260 agcaacacca ccgagatctt ccgccccggc ggcggcgaca tgcgcgacaa ctggcgcagc 1320 gagctgtaca agtacaaggt ggtgaagatc gagcccctgg gcgtggcccc caccatcgcc 1380 atcagcagcg tggtgcagag cgagaagagc gccgtgaccc tgggcgccat gttcctgggc 1440 ttcctgggcg ccgccggcag caccatgggc gcccgcagcc tgaccctgac cgtgcaggcc 1500 cgccagctgc tgagcggcat cgtgcagcag cagaacaacc tgctgcgcgc catcgaggcc 1560 cagcagcacc tgctgcagct gaccgtgtgg ggcatcaagc agctgcaggc ccgcgtgctg 1620 gccgtggagc gctacctgaa ggaccagcag ctgctgggca tctggggctg cagcggcaag 1680 ctgatctgca ccaccgccgt gccctggaac gccagctgga gcaacaagag cctggaccag 1740 atctggaaca acatgacctg gatggagtgg gagcgcgaga tcgacaacta caccaacctg 1800 atctacaccc tgatcgagga gagccagaac cagcaggaga agaacgagca ggagctgctg 1860 gagctggaca agtgggccag cctgtggaac tggttcgaca tcagcaagtg gctgtggtac 1920 atcaagatct tcatcatgat cgtgggcggc ctggtgggcc tgcgcatcgt gttcaccgtg 1980 ctgagcatcg tgaaccgcgt gcgccagggc tacagccccc tgagcttcca gacccgcttc 2040 cccgcccccc gcggccccga ccgccccgag ggcatcgagg aggagggcgg cgagcgcgac 2100 cgcgaccgca gcagccccct ggtgcacggc ctgctggccc tgatctggga cgacctgcgc 2160 agcctgtgcc tgttcagcta ccaccgcctg cgcgacctga tcctgatcgc cgcccgcatc 2220 gtggagctgc tgggccgccg cggctgggag gccctgaagt actggggcaa cctgctgcag 2280 tactggatcc aggagctgaa gaacagcgcc gtgagcctgt tcgacgccat cgccatcgcc 2340 gtggccgagg gcaccgaccg catcatcgag gtggcccagc gcatcggccg cgccttcctg 2400 cacatccccc gccgcatccg ccagggcttc gagcgcgccc tgctgtaagr sht 2453 23 866 DNA Artificial Sequence Description of Artificial Sequence int.opt.mut.SF2 23 ttcctgaacg gcatcgacaa ggcccaggag gagcacgaga agtaccacag caactggcgc 60 gccatggcca gcgacttcaa cctgcccccc gtggtggcca aggagatcgt ggccagcgcc 120 gacaagtgcc agctgaaggg cgaggccatg cacggccagg tggactgcag ccccggcatc 180 tggcagctgg cctgcaccca cctggagggc aagatcatcc tggtggccgt gcacgtggcc 240 agcggctaca tcgaggccga ggtgatcccc gccgagaccg gccaggagac cgcctacttc 300 ctgctgaagc tggccggccg ctggcccgtg aagaccatcc acaccgccaa cggcagcaac 360 ttcaccagca ccaccgtgaa ggccgcctgc tggtgggccg gcatcaagca ggagttcggc 420 atcccctaca acccccagag ccagggcgtg gtggcgagca tgaacaacga gctgaagaag 480 atcatcggcc aggtgcgcga ccaggccgag cacctgaaga ccgccgtgca gatggccgtg 540 ttcatccaca acttcaagcg caagggcggc atcggcggct acagcgccgg cgagcgcatc 600 gtggacatca tcgccaccga catccagacc aaggagctgc agaagcagat caccaagatc 660 cagaacttcc gcgtgtacta ccgcgacaac aaggaccccc tgaagggccc cgccaagctg 720 ctgtggaagg gcgagggcgc cgtggtgatc caggacaaca gcgacatcaa ggtggtgccc 780 cgccgcaagg ccaagatcat ccgcgactac ggcaagcaga tggccggcga cgactgcgtg 840 gccagccgcc aggacgagga cgrsht 866 24 869 DNA Artificial Sequence Description of Artificial Sequence int.opt.SF2 24 ttcctgaacg gcatcgacaa ggcccaggag gagcacgaga agtaccacag caactggcgc 60 gccatggcca gcgacttcaa cctgcccccc gtggtggcca aggagatcgt ggccagctgc 120 gacaagtgcc agctgaaggg cgaggccatg cacggccagg tggactgcag ccccggcatc 180 tggcagctgg actgcaccca cctggagggc aagatcatcc tggtggccgt gcacgtggcc 240 agcggctaca tcgaggccga ggtgatcccc gccgagaccg gccaggagac cgcctacttc 300 ctgctgaagc tggccggccg ctggcccgtg aagaccatcc acaccgacaa cggcagcaac 360 ttcaccagca ccaccgtgaa ggccgcctgc tggtgggccg gcatcaagca ggagttcggc 420 atcccctaca acccccagag ccagggcgtg gtggagagca tgaacaacga gctgaagaag 480 atcatcggcc aggtgcgcga ccaggccgag cacctgaaga ccgccgtgca gatggccgtg 540 ttcatccaca acttcaagcg caagggcggc atcggcggct acagcgccgg cgagcgcatc 600 gtggacatca tcgccaccga catccagacc aaggagctgc agaagcagat caccaagatc 660 cagaacttcc gcgtgtacta ccgcgacaac aaggaccccc tgtggaaggg ccccgccaag 720 ctgctgtgga agggcgaggg cgccgtggtg atccaggaca acagcgacat caaggtggtg 780 ccccgccgca aggccaagat catccgcgac tacggcaagc agatggccgg cgacgactgc 840 gtggccagcc gccaggacga ggacgrsht 869 25 629 DNA Artificial Sequence Description of Artificial Sequence nef.D125G.-myr.opt.SF162 25 atggccggca agtggagcaa gcgcatgagc ggctggagcg ccgtgcgcga gcgcatgaag 60 cgcgccgagc ccgccgagcc cgccgccgac ggcgtgggcg ccgtgagccg cgacctggag 120 aagcacggcg ccatcaccag cagcaacacc gccgccaaca acgccgactg cgcctggctg 180 gaggcccagg aggacgagga cgtgggcttc cccgtgcgcc cccaggtgcc cctgcgcccc 240 atgacctaca aggccgccct ggacctgagc cacttcctga aggagaaggg cggcctggag 300 ggcctgatct acagccagaa gcgccaggac atcctggacc tgtggatcca ccacacccag 360 ggctacttcc ccggctggca gaactacacc cccggccccg gcatccgcta ccccctgacc 420 ttcggctggt gcttcaagct ggtgcccgtg gaccccgact acgtggagga ggccaacgcc 480 ggcgagaaca acagcctgct gcaccccatg agccagcacg gcatggacga ccccgagaag 540 gaggtgctgg tgtggcgctt cgacagccgc ctggccttcc accacatggc ccgcgagctg 600 caccccgagt actacaagga ctgcgrsht 629 26 570 DNA Artificial Sequence Description of Artificial Sequence nef.D107G.-myr18.opt.SF162 26 atgaagcgcg ccgagcccgc cgagcccgcc gccgacggcg tgggcgccgt gagccgcgac 60 ctggagaagc acggcgccat caccagcagc aacaccgccg ccaacaacgc cgactgcgcc 120 tggctggagg cccaggagga cgaggacgtg ggcttccccg tgcgccccca ggtgcccctg 180 cgccccatga cctacaaggc cgccctggac ctgagccact tcctgaagga gaagggcggc 240 ctggagggcc tgatctacag ccagaagcgc caggacatcc tggacctgtg gatccaccac 300 acccagggct acttccccgg ctggcagaac tacacccccg gccccggcat ccgctacccc 360 ctgaccttcg gctggtgctt caagctggtg cccgtggacc ccgactacgt ggaggaggcc 420 aacgccggcg agaacaacag cctgctgcac cccatgagcc agcacggcat ggacgacccc 480 gagaaggagg tgctggtgtg gcgcttcgac agccgcctgg ccttccacca catggcccgc 540 gagctgcacc ccgagtacta caaggactgc 570 27 624 DNA Artificial Sequence Description of Artificial Sequence nef.opt.D125G.SF162 27 atgggcggca agtggagcaa gcgcatgagc ggctggagcg ccgtgcgcga gcgcatgaag 60 cgcgccgagc ccgccgagcc cgccgccgac ggcgtgggcg ccgtgagccg cgacctggag 120 aagcacggcg ccatcaccag cagcaacacc gccgccaaca acgccgactg cgcctggctg 180 gaggcccagg aggacgagga cgtgggcttc cccgtgcgcc cccaggtgcc cctgcgcccc 240 atgacctaca aggccgccct ggacctgagc cacttcctga aggagaaggg cggcctggag 300 ggcctgatct acagccagaa gcgccaggac atcctggacc tgtggatcca ccacacccag 360 ggctacttcc ccggctggca gaactacacc cccggccccg gcatccgcta ccccctgacc 420 ttcggctggt gcttcaagct ggtgcccgtg gaccccgact acgtggagga ggccaacgcc 480 ggcgagaaca acagcctgct gcaccccatg agccagcacg gcatggacga ccccgagaag 540 gaggtgctgg tgtggcgctt cgacagccgc ctggccttcc accacatggc ccgcgagctg 600 caccccgagt actacaagga ctgc 624 28 624 DNA Artificial Sequence Description of Artificial Sequence nef.opt. SF162 28 atgggcggca agtggagcaa gcgcatgagc ggctggagcg ccgtgcgcga gcgcatgaag 60 cgcgccgagc ccgccgagcc cgccgccgac ggcgtgggcg ccgtgagccg cgacctggag 120 aagcacggcg ccatcaccag cagcaacacc gccgccaaca acgccgactg cgcctggctg 180 gaggcccagg aggacgagga

cgtgggcttc cccgtgcgcc cccaggtgcc cctgcgcccc 240 atgacctaca aggccgccct ggacctgagc cacttcctga aggagaaggg cggcctggag 300 ggcctgatct acagccagaa gcgccaggac atcctggacc tgtggatcca ccacacccag 360 ggctacttcc ccgactggca gaactacacc cccggccccg gcatccgcta ccccctgacc 420 ttcggctggt gcttcaagct ggtgcccgtg gaccccgact acgtggagga ggccaacgcc 480 ggcgagaaca acagcctgct gcaccccatg agccagcacg gcatggacga ccccgagaag 540 gaggtgctgg tgtggcgctt cgacagccgc ctggccttcc accacatggc ccgcgagctg 600 caccccgagt actacaagga ctgc 624 29 360 DNA Artificial Sequence Description of Artificial Sequence p15RnaseH.opt.SF2 29 tacgtggacg gcgccgccaa ccgcgagacc aagctgggca aggccggcta cgtgaccgac 60 cggggccggc agaaggtggt gagcatcgcc gacaccacca accagaagac cgagctgcag 120 gccatccacc tggccctgca ggacagcggc ctggaggtga acatcgtgac cgacagccag 180 tacgccctgg gcatcatcca ggcccagccc gacaagagcg agagcgagct ggtgagccag 240 atcatcgagc agctgatcaa gaaggagaag gtgtacctgg cctgggtgcc cgcccacaag 300 ggcatcggcg gcaacgagca ggtggacaag ctggtgagcg ccggcatccg caaggtgctg 360 30 2460 DNA Artificial Sequence Description of Artificial Sequence p2Pol.opt.YMWM.SF2 30 gccaccatgg ccgaggcgat gagccaggtg acgaacccgg cgaccatcat gatgcagcgc 60 ggcaacttcc gcaaccagcg gaagaccgtc aagtgcttca actgcggcaa ggagggccac 120 accgccagga actgccgcgc cccccgcaag aagggctgct ggcgctgcgg ccgcgaagga 180 caccaaatga aagattgcac tgagagacag gctaatttct tccgcgagga cctggccttc 240 ctgcagggca aggcccgcga gttcagcagc gagcagaccc gcgccaacag ccccacccgc 300 cgcgagctgc aggtgtgggg cggcgagaac aacagcctga gcgaggccgg cgccgaccgc 360 cagggcaccg tgagcttcaa cttcccccag atcaccctgt ggcagcgccc cctggtgacc 420 atcaggatcg gcggccagct caaggaggcg ctgctcgcca ccggcgccga cgacaccgtg 480 ctggaggaga tgaacctgcc cggcaagtgg aagcccaaga tgatcggcgg gatcgggggc 540 ttcatcaagg tgcggcagta cgaccagatc cccgtggaga tctgcggcca caaggccatc 600 ggcaccgtgc tggtgggccc cacccccgtg aacatcatcg gccgcaacct gctgacccag 660 atcggctgca ccctgaactt ccccatcagc cccatcgaga cggtgcccgt gaagctgaag 720 ccggggatgg acggccccaa ggtcaagcag tggcccctga ccgaggagaa gatcaaggcc 780 ctggtggaga tctgcaccga gatggagaag gagggcaaga tcagcaagat cggccccgag 840 aacccctaca acacccccgt gttcgccatc aagaagaagg acagcaccaa gtggcgcaag 900 ctggtggact tccgcgagct gaacaagcgc acccaggact tctgggaggt gcagctgggc 960 atcccccacc ccgccggcct gaagaagaag aagagcgtga ccgtgctgga cgtgggcgac 1020 gcctacttca gcgtgcccct ggacaaggac ttccgcaagt acaccgcctt caccatcccc 1080 agcatcaaca acgagacccc cggcatccgc taccagtaca acgtgctgcc ccagggctgg 1140 aagggcagcc ccgccatctt ccagagcagc atgaccaaga tcctggagcc cttccgcaag 1200 cagaaccccg acatcgtgat ctaccaggcc cccctgtacg tgggcagcga cctggagatc 1260 ggccagcacc gcaccaagat cgaggagctg cgccagcacc tgctgcgctg gggcttcacc 1320 acccccgaca agaagcacca gaaggagccc cccttcctgc ccatcgagct gcaccccgac 1380 aagtggaccg tgcagcccat catgctgccc gagaaggaca gctggaccgt gaacgacatc 1440 cagaagctgg tgggcaagct gaactgggcc agccagatct acgccggcat caaggtgaag 1500 cagctgtgca agctgctgcg cggcaccaag gccctgaccg aggtgatccc cctgaccgag 1560 gaggccgagc tggagctggc cgagaaccgc gagatcctga aggagcccgt gcacgaggtg 1620 tactacgacc ccagcaagga cctggtggcc gagatccaga agcagggcca gggccagtgg 1680 acctaccaga tctaccagga gcccttcaag aacctgaaga ccggcaagta cgcccgcatg 1740 cgcggcgccc acaccaacga cgtgaagcag ctgaccgagg ccgtgcagaa ggtgagcacc 1800 gagagcatcg tgatctgggg caagatcccc aagttcaagc tgcccatcca gaaggagacc 1860 tgggaggcct ggtggatgga gtactggcag gccacctgga tccccgagtg ggagttcgtg 1920 aacacccccc ccctggtgaa gctgtggtac cagctggaga aggagcccat cgtgggcgcc 1980 gagaccttct acgtggacgg cgccgccaac cgcgagacca agctgggcaa ggccggctac 2040 gtgaccgacc ggggccggca gaaggtggtg agcatcgccg acaccaccaa ccagaagacc 2100 gagctgcagg ccatccacct ggccctgcag gacagcggcc tggaggtgaa catcgtgacc 2160 gacagccagt acgccctggg catcatccag gcccagcccg acaagagcga gagcgagctg 2220 gtgagccaga tcatcgagca gctgatcaag aaggagaagg tgtacctggc ctgggtgccc 2280 gcccacaagg gcatcggcgg caacgagcag gtggacaagc tggtgagcgc cggcatccgc 2340 aaggtgctgt tcctgaacgg catcgatggc ggcatcgtga tctaccagta catggacgac 2400 ctgtacgtgg gcagcggcgg ccctaggatc gattaaaagc ttcccggggc tagcaccggt 2460 31 2466 DNA Artificial Sequence Description of Artificial Sequence p2PolInaopt.YM.SF2 31 gccaccatgg ccgaggcgat gagccaggtg acgaacccgg cgaccatcat gatgcagcgc 60 ggcaacttcc gcaaccagcg gaagaccgtc aagtgcttca actgcggcaa ggagggccac 120 accgccagga actgccgcgc cccccgcaag aagggctgct ggcgctgcgg ccgcgaagga 180 caccaaatga aagattgcac tgagagacag gctaatttct tccgcgagga cctggccttc 240 ctgcagggca aggcccgcga gttcagcagc gagcagaccc gcgccaacag ccccacccgc 300 cgcgagctgc aggtgtgggg cggcgagaac aacagcctga gcgaggccgg cgccgaccgc 360 cagggcaccg tgagcttcaa cttcccccag atcaccctgt ggcagcgccc cctggtgacc 420 atcaggatcg gcggccagct caaggaggcg ctgctcgcca ccggcgccga cgacaccgtg 480 ctggaggaga tgaacctgcc cggcaagtgg aagcccaaga tgatcggcgg gatcgggggc 540 ttcatcaagg tgcggcagta cgaccagatc cccgtggaga tctgcggcca caaggccatc 600 ggcaccgtgc tggtgggccc cacccccgtg aacatcatcg gccgcaacct gctgacccag 660 atcggctgca ccctgaactt ccccatcagc cccatcgaga cggtgcccgt gaagctgaag 720 ccggggatgg acggccccaa ggtcaagcag tggcccctga ccgaggagaa gatcaaggcc 780 ctggtggaga tctgcaccga gatggagaag gagggcaaga tcagcaagat cggccccgag 840 aacccctaca acacccccgt gttcgccatc aagaagaagg acagcaccaa gtggcgcaag 900 ctggtggact tccgcgagct gaacaagcgc acccaggact tctgggaggt gcagctgggc 960 atcccccacc ccgccggcct gaagaagaag aagagcgtga ccgtgctgga cgtgggcgac 1020 gcctacttca gcgtgcccct ggacaaggac ttccgcaagt acaccgcctt caccatcccc 1080 agcatcaaca acgagacccc cggcatccgc taccagtaca acgtgctgcc ccagggctgg 1140 aagggcagcc ccgccatctt ccagagcagc atgaccaaga tcctggagcc cttccgcaag 1200 cagaaccccg acatcgtgat ctaccaggcc cccctgtacg tgggcagcga cctggagatc 1260 ggccagcacc gcaccaagat cgaggagctg cgccagcacc tgctgcgctg gggcttcacc 1320 acccccgaca agaagcacca gaaggagccc cccttcctgt ggatgggcta cgagctgcac 1380 cccgacaagt ggaccgtgca gcccatcatg ctgcccgaga aggacagctg gaccgtgaac 1440 gacatccaga agctggtggg caagctgaac tgggccagcc agatctacgc cggcatcaag 1500 gtgaagcagc tgtgcaagct gctgcgcggc accaaggccc tgaccgaggt gatccccctg 1560 accgaggagg ccgagctgga gctggccgag aaccgcgaga tcctgaagga gcccgtgcac 1620 gaggtgtact acgaccccag caaggacctg gtggccgaga tccagaagca gggccagggc 1680 cagtggacct accagatcta ccaggagccc ttcaagaacc tgaagaccgg caagtacgcc 1740 cgcatgcgcg gcgcccacac caacgacgtg aagcagctga ccgaggccgt gcagaaggtg 1800 agcaccgaga gcatcgtgat ctggggcaag atccccaagt tcaagctgcc catccagaag 1860 gagacctggg aggcctggtg gatggagtac tggcaggcca cctggatccc cgagtgggag 1920 ttcgtgaaca ccccccccct ggtgaagctg tggtaccagc tggagaagga gcccatcgtg 1980 ggcgccgaga ccttctacgt ggacggcgcc gccaaccgcg agaccaagct gggcaaggcc 2040 ggctacgtga ccgaccgggg ccggcagaag gtggtgagca tcgccgacac caccaaccag 2100 aagaccgagc tgcaggccat ccacctggcc ctgcaggaca gcggcctgga ggtgaacatc 2160 gtgaccgaca gccagtacgc cctgggcatc atccaggccc agcccgacaa gagcgagagc 2220 gagctggtga gccagatcat cgagcagctg atcaagaagg agaaggtgta cctggcctgg 2280 gtgcccgccc acaagggcat cggcggcaac gagcaggtgg acaagctggt gagcgccggc 2340 atccgcaagg tgctgttcct gaacggcatc gatggcggca tcgtgatcta ccagtacatg 2400 gacgacctgt acgtgggcag cggcggccct aggatcgatt aaaagcttcc cggggctagc 2460 accggt 2466 32 2472 DNA Artificial Sequence Description of Artificial Sequence p2Polopt.SF2 32 gccaccatgg ccgaggcgat gagccaggtg acgaacccgg cgaccatcat gatgcagcgc 60 ggcaacttcc gcaaccagcg gaagaccgtc aagtgcttca actgcggcaa ggagggccac 120 accgccagga actgccgcgc cccccgcaag aagggctgct ggcgctgcgg ccgcgaagga 180 caccaaatga aagattgcac tgagagacag gctaatttct tccgcgagga cctggccttc 240 ctgcagggca aggcccgcga gttcagcagc gagcagaccc gcgccaacag ccccacccgc 300 cgcgagctgc aggtgtgggg cggcgagaac aacagcctga gcgaggccgg cgccgaccgc 360 cagggcaccg tgagcttcaa cttcccccag atcaccctgt ggcagcgccc cctggtgacc 420 atcaggatcg gcggccagct caaggaggcg ctgctcgaca ccggcgccga cgacaccgtg 480 ctggaggaga tgaacctgcc cggcaagtgg aagcccaaga tgatcggcgg gatcgggggc 540 ttcatcaagg tgcggcagta cgaccagatc cccgtggaga tctgcggcca caaggccatc 600 ggcaccgtgc tggtgggccc cacccccgtg aacatcatcg gccgcaacct gctgacccag 660 atcggctgca ccctgaactt ccccatcagc cccatcgaga cggtgcccgt gaagctgaag 720 ccggggatgg acggccccaa ggtcaagcag tggcccctga ccgaggagaa gatcaaggcc 780 ctggtggaga tctgcaccga gatggagaag gagggcaaga tcagcaagat cggccccgag 840 aacccctaca acacccccgt gttcgccatc aagaagaagg acagcaccaa gtggcgcaag 900 ctggtggact tccgcgagct gaacaagcgc acccaggact tctgggaggt gcagctgggc 960 atcccccacc ccgccggcct gaagaagaag aagagcgtga ccgtgctgga cgtgggcgac 1020 gcctacttca gcgtgcccct ggacaaggac ttccgcaagt acaccgcctt caccatcccc 1080 agcatcaaca acgagacccc cggcatccgc taccagtaca acgtgctgcc ccagggctgg 1140 aagggcagcc ccgccatctt ccagagcagc atgaccaaga tcctggagcc cttccgcaag 1200 cagaaccccg acatcgtgat ctaccagtac atggacgacc tgtacgtggg cagcgacctg 1260 gagatcggcc agcaccgcac caagatcgag gagctgcgcc agcacctgct gcgctggggc 1320 ttcaccaccc ccgacaagaa gcaccagaag gagcccccct tcctgtggat gggctacgag 1380 ctgcaccccg acaagtggac cgtgcagccc atcatgctgc ccgagaagga cagctggacc 1440 gtgaacgaca tccagaagct ggtgggcaag ctgaactggg ccagccagat ctacgccggc 1500 atcaaggtga agcagctgtg caagctgctg cgcggcacca aggccctgac cgaggtgatc 1560 cccctgaccg aggaggccga gctggagctg gccgagaacc gcgagatcct gaaggagccc 1620 gtgcacgagg tgtactacga ccccagcaag gacctggtgg ccgagatcca gaagcagggc 1680 cagggccagt ggacctacca gatctaccag gagcccttca agaacctgaa gaccggcaag 1740 tacgcccgca tgcgcggcgc ccacaccaac gacgtgaagc agctgaccga ggccgtgcag 1800 aaggtgagca ccgagagcat cgtgatctgg ggcaagatcc ccaagttcaa gctgcccatc 1860 cagaaggaga cctgggaggc ctggtggatg gagtactggc aggccacctg gatccccgag 1920 tgggagttcg tgaacacccc ccccctggtg aagctgtggt accagctgga gaaggagccc 1980 atcgtgggcg ccgagacctt ctacgtggac ggcgccgcca accgcgagac caagctgggc 2040 aaggccggct acgtgaccga ccggggccgg cagaaggtgg tgagcatcgc cgacaccacc 2100 aaccagaaga ccgagctgca ggccatccac ctggccctgc aggacagcgg cctggaggtg 2160 aacatcgtga ccgacagcca gtacgccctg ggcatcatcc aggcccagcc cgacaagagc 2220 gagagcgagc tggtgagcca gatcatcgag cagctgatca agaaggagaa ggtgtacctg 2280 gcctgggtgc ccgcccacaa gggcatcggc ggcaacgagc aggtggacaa gctggtgagc 2340 gccggcatcc gcaaggtgct gttcctgaac ggcatcgatg gcggcatcgt gatctaccag 2400 tacatggacg acctgtacgt gggcagcggc ggccctagga tcgattaaaa gcttcccggg 2460 gctagcaccg gt 2472 33 3639 DNA Artificial Sequence Description of Artificial Sequence p2PolTatRevNef.opt.native_B 33 atggccgagg cgatgagcca ggtgacgaac ccggcgacca tcatgatgca gcgcggcaac 60 ttccgcaacc agcggaagac cgtcaagtgc ttcaactgcg gcaaggaggg ccacaccgcc 120 aggaactgcc gcgccccccg caagaagggc tgctggcgct gcggccgcga aggacaccaa 180 atgaaagatt gcactgagag acaggctaat ttcttccgcg aggacctggc cttcctgcag 240 ggcaaggccc gcgagttcag cagcgagcag acccgcgcca acagccccac ccgccgcgag 300 ctgcaggtgt ggggcggcga gaacaacagc ctgagcgagg ccggcgccga ccgccagggc 360 accgtgagct tcaacttccc ccagatcacc ctgtggcagc gccccctggt gaccatcagg 420 atcggcggcc agctcaagga ggcgctgctc gacaccggcg ccgacgacac cgtgctggag 480 gagatgaacc tgcccggcaa gtggaagccc aagatgatcg gcgggatcgg gggcttcatc 540 aaggtgcggc agtacgacca gatccccgtg gagatctgcg gccacaaggc catcggcacc 600 gtgctggtgg gccccacccc cgtgaacatc atcggccgca acctgctgac ccagatcggc 660 tgcaccctga acttccccat cagccccatc gagacggtgc ccgtgaagct gaagccgggg 720 atggacggcc ccaaggtcaa gcagtggccc ctgaccgagg agaagatcaa ggccctggtg 780 gagatctgca ccgagatgga gaaggagggc aagatcagca agatcggccc cgagaacccc 840 tacaacaccc ccgtgttcgc catcaagaag aaggacagca ccaagtggcg caagctggtg 900 gacttccgcg agctgaacaa gcgcacccag gacttctggg aggtgcagct gggcatcccc 960 caccccgccg gcctgaagaa gaagaagagc gtgaccgtgc tggacgtggg cgacgcctac 1020 ttcagcgtgc ccctggacaa ggacttccgc aagtacaccg ccttcaccat ccccagcatc 1080 aacaacgaga cccccggcat ccgctaccag tacaacgtgc tgccccaggg ctggaagggc 1140 agccccgcca tcttccagag cagcatgacc aagatcctgg agcccttccg caagcagaac 1200 cccgacatcg tgatctacca gtacatggac gacctgtacg tgggcagcga cctggagatc 1260 ggccagcacc gcaccaagat cgaggagctg cgccagcacc tgctgcgctg gggcttcacc 1320 acccccgaca agaagcacca gaaggagccc cccttcctgt ggatgggcta cgagctgcac 1380 cccgacaagt ggaccgtgca gcccatcatg ctgcccgaga aggacagctg gaccgtgaac 1440 gacatccaga agctggtggg caagctgaac tgggccagcc agatctacgc cggcatcaag 1500 gtgaagcagc tgtgcaagct gctgcgcggc accaaggccc tgaccgaggt gatccccctg 1560 accgaggagg ccgagctgga gctggccgag aaccgcgaga tcctgaagga gcccgtgcac 1620 gaggtgtact acgaccccag caaggacctg gtggccgaga tccagaagca gggccagggc 1680 cagtggacct accagatcta ccaggagccc ttcaagaacc tgaagaccgg caagtacgcc 1740 cgcatgcgcg gcgcccacac caacgacgtg aagcagctga ccgaggccgt gcagaaggtg 1800 agcaccgaga gcatcgtgat ctggggcaag atccccaagt tcaagctgcc catccagaag 1860 gagacctggg aggcctggtg gatggagtac tggcaggcca cctggatccc cgagtgggag 1920 ttcgtgaaca ccccccccct ggtgaagctg tggtaccagc tggagaagga gcccatcgtg 1980 ggcgccgaga ccttctacgt ggacggcgcc gccaaccgcg agaccaagct gggcaaggcc 2040 ggctacgtga ccgaccgggg ccggcagaag gtggtgagca tcgccgacac caccaaccag 2100 aagaccgagc tgcaggccat ccacctggcc ctgcaggaca gcggcctgga ggtgaacatc 2160 gtgaccgaca gccagtacgc cctgggcatc atccaggccc agcccgacaa gagcgagagc 2220 gagctggtga gccagatcat cgagcagctg atcaagaagg agaaggtgta cctggcctgg 2280 gtgcccgccc acaagggcat cggcggcaac gagcaggtgg acaagctggt gagcgccggc 2340 atccgcaagg tgctggaatt cgagcccgtg gacccccgcc tggagccctg gaagcacccc 2400 ggcagccagc ccaagaccgc ctgcaccaac tgctactgca agaagtgctg cttccactgc 2460 caggtgtgct tcatcaccaa gggcctgggc atcagctacg gccgcaagaa gcgccgccag 2520 cgccgccgcg ccccccccga cagcgaggtg caccaggtga gcctgcccaa gcagcccgcc 2580 agccagcccc agggcgaccc caccggcccc aaggagagca agaagaaggt ggagcgcgag 2640 accgagaccg accccgtgca ccccggggcc ggccgcagcg gcgacagcga cgaggagctg 2700 ctgcagaccg tgcgcttcat caagttcctg taccagagca accccctgcc cagccccaag 2760 ggcacccgcc aggcccgccg caaccgccgc cgccgctggc gcgagcgcca gcgccagatc 2820 cagagcatca gcgcctggat catcagcacc cacctgggcc gcagcaccga gcccgtgccc 2880 ctgcagctgc cccccctgga gcgcctgaac ctggactgca gcgaggactg cggcaccagc 2940 ggcacccagg gcgtgggcag cccccaggtg ctgggcgaga gccccgccgt gctggacagc 3000 ggcaccaagg agctcgaggg cggcaagtgg agcaagcgca tgagcggctg gagcgccgtg 3060 cgcgagcgca tgaagcgcgc cgagcccgcc gagcccgccg ccgacggcgt gggcgccgtg 3120 agccgcgacc tggagaagca cggcgccatc accagcagca acaccgccgc caacaacgcc 3180 gactgcgcct ggctggaggc ccaggaggac gaggacgtgg gcttccccgt gcgcccccag 3240 gtgcccctgc gccccatgac ctacaaggcc gccctggacc tgagccactt cctgaaggag 3300 aagggcggcc tggagggcct gatctacagc cagaagcgcc aggacatcct ggacctgtgg 3360 atccaccaca cccagggcta cttccccgac tggcagaact acacccccgg ccccggcatc 3420 cgctaccccc tgaccttcgg ctggtgcttc aagctggtgc ccgtggaccc cgactacgtg 3480 gaggaggcca acgccggcga gaacaacagc ctgctgcacc ccatgagcca gcacggcatg 3540 gacgaccccg agaaggaggt gctggtgtgg cgcttcgaca gccgcctggc cttccaccac 3600 atggcccgcg agctgcaccc cgagtactac aaggactgc 3639 34 3735 DNA Artificial Sequence Description of Artificial Sequence p2PolTatRevNef.opt_B 34 gccaccatgg ccgaggcgat gagccaggtg acgaacccgg cgaccatcat gatgcagcgc 60 ggcaacttcc gcaaccagcg gaagaccgtc aagtgcttca actgcggcaa ggagggccac 120 accgccagga actgccgcgc cccccgcaag aagggctgct ggcgctgcgg ccgcgaagga 180 caccaaatga aagattgcac tgagagacag gctaatttct tccgcgagga cctggccttc 240 ctgcagggca aggcccgcga gttcagcagc gagcagaccc gcgccaacag ccccacccgc 300 cgcgagctgc aggtgtgggg cggcgagaac aacagcctga gcgaggccgg cgccgaccgc 360 cagggcaccg tgagcttcaa cttcccccag atcaccctgt ggcagcgccc cctggtgacc 420 atcaggatcg gcggccagct caaggaggcg ctgctcgcca ccggcgccga cgacaccgtg 480 ctggaggaga tgaacctgcc cggcaagtgg aagcccaaga tgatcggcgg gatcgggggc 540 ttcatcaagg tgcggcagta cgaccagatc cccgtggaga tctgcggcca caaggccatc 600 ggcaccgtgc tggtgggccc cacccccgtg aacatcatcg gccgcaacct gctgacccag 660 atcggctgca ccctgaactt ccccatcagc cccatcgaga cggtgcccgt gaagctgaag 720 ccggggatgg acggccccaa ggtcaagcag tggcccctga ccgaggagaa gatcaaggcc 780 ctggtggaga tctgcaccga gatggagaag gagggcaaga tcagcaagat cggccccgag 840 aacccctaca acacccccgt gttcgccatc aagaagaagg acagcaccaa gtggcgcaag 900 ctggtggact tccgcgagct gaacaagcgc acccaggact tctgggaggt gcagctgggc 960 atcccccacc ccgccggcct gaagaagaag aagagcgtga ccgtgctgga cgtgggcgac 1020 gcctacttca gcgtgcccct ggacaaggac ttccgcaagt acaccgcctt caccatcccc 1080 agcatcaaca acgagacccc cggcatccgc taccagtaca acgtgctgcc ccagggctgg 1140 aagggcagcc ccgccatctt ccagagcagc atgaccaaga tcctggagcc cttccgcaag 1200 cagaaccccg acatcgtgat ctaccaggcc cccctgtacg tgggcagcga cctggagatc 1260 ggccagcacc gcaccaagat cgaggagctg cgccagcacc tgctgcgctg gggcttcacc 1320 acccccgaca agaagcacca gaaggagccc cccttcctgc ccatcgagct gcaccccgac 1380 aagtggaccg tgcagcccat catgctgccc gagaaggaca gctggaccgt gaacgacatc 1440 cagaagctgg tgggcaagct gaactgggcc agccagatct acgccggcat caaggtgaag 1500 cagctgtgca agctgctgcg cggcaccaag gccctgaccg aggtgatccc cctgaccgag 1560 gaggccgagc tggagctggc cgagaaccgc gagatcctga aggagcccgt gcacgaggtg 1620 tactacgacc ccagcaagga cctggtggcc gagatccaga agcagggcca gggccagtgg 1680 acctaccaga tctaccagga gcccttcaag aacctgaaga ccggcaagta cgcccgcatg 1740 cgcggcgccc acaccaacga cgtgaagcag ctgaccgagg ccgtgcagaa ggtgagcacc 1800 gagagcatcg tgatctgggg caagatcccc aagttcaagc tgcccatcca gaaggagacc 1860 tgggaggcct ggtggatgga gtactggcag gccacctgga tccccgagtg ggagttcgtg 1920 aacacccccc ccctggtgaa gctgtggtac cagctggaga aggagcccat cgtgggcgcc 1980 gagaccttct acgtggacgg cgccgccaac cgcgagacca agctgggcaa ggccggctac 2040 gtgaccgacc ggggccggca gaaggtggtg agcatcgccg acaccaccaa ccagaagacc 2100 gagctgcagg ccatccacct ggccctgcag gacagcggcc tggaggtgaa catcgtgacc 2160 gacagccagt acgccctggg catcatccag gcccagcccg acaagagcga gagcgagctg 2220 gtgagccaga tcatcgagca gctgatcaag aaggagaagg tgtacctggc ctgggtgccc 2280 gcccacaagg gcatcggcgg caacgagcag gtggacaagc tggtgagcgc cggcatccgc 2340 aaggtgctgt tcctgaacgg catcgatggc ggcatcgtga tctaccagta catggacgac 2400 ctgtacgtgg gcagcggcgg ccctagggag cccgtggacc cccgcctgga gccctggaag 2460 caccccggca gccagcccaa gaccgccggc accaactgct actgcaagaa gtgctgcttc 2520 cactgccagg tgagcttcat caccaagggc ctgggcatca gctacggccg caagaagcgc 2580

cgccagcgcc gccgcgcccc ccccgacagc gaggtgcacc aggtgagcct gcccaagcag 2640 cccgccagcc agccccaggg cgaccccacc ggccccaagg agagcaagaa gaaggtggag 2700 cgcgagaccg agaccgaccc cgtgcacccc ggggccggcc gcagcggcga cagcgacgag 2760 gagctgctgc agaccgtgcg cttcatcaag ttcctgtacc agagcaaccc cctgcccagc 2820 cccaagggca cccgccaggc cgacctgaac cgccgccgcc gctggcgcga gcgccagcgc 2880 cagatccaga gcatcagcgc ctggatcatc agcacccacc tgggccgcag caccgagccc 2940 gtgcccctgc agctgccccc cgacctgcgc ctgaacctgg actgcagcga ggactgcggc 3000 accagcggca cccagggcgt gggcagcccc caggtgctgg gcgagagccc cgccgtgctg 3060 gacagcggca ccaaggagct cgaggccggc aagtggagca agcgcatgag cggctggagc 3120 gccgtgcgcg agcgcatgaa gcgcgccgag cccgccgagc ccgccgccga cggcgtgggc 3180 gccgtgagcc gcgacctgga gaagcacggc gccatcacca gcagcaacac cgccgccaac 3240 aacgccgact gcgcctggct ggaggcccag gaggacgagg acgtgggctt ccccgtgcgc 3300 ccccaggtgc ccctgcgccc catgacctac aaggccgccc tggacctgag ccacttcctg 3360 aaggagaagg gcggcctgga gggcctgatc tacagccaga agcgccagga catcctggac 3420 ctgtggatcc accacaccca gggctacttc cccggctggc agaactacac ccccggcccc 3480 ggcatccgct accccctgac cttcggctgg tgcttcaagc tggtgcccgt ggaccccgac 3540 tacgtggagg aggccaacgc cggcgagaac aacagcctgc tgcaccccat gagccagcac 3600 ggcatggacg accccgagaa ggaggtgctg gtgtggcgct tcgacagccg cctggccttc 3660 caccacatgg cccgcgagct gcaccccgag tactacaagg actgcgatta aaagcttccc 3720 ggggctagca ccggt 3735 35 2145 DNA Artificial Sequence Description of Artificial Sequence pol.opt.SF2 35 ttcttccgcg aggacctggc cttcctgcag ggcaaggccc gcgagttcag cagcgagcag 60 acccgcgcca acagccccac ccgccgcgag ctgcaggtgt ggggcggcga gaacaacagc 120 ctgagcgagg ccggcgccga ccgccagggc accgtgagct tcaacttccc ccagatcacc 180 ctgtggcagc gccccctggt gaccatcagg atcggcggcc agctcaagga ggcgctgctc 240 gacaccggcg ccgacgacac cgtgctggag gagatgaacc tgcccggcaa gtggaagccc 300 aagatgatcg gcgggatcgg gggcttcatc aaggtgcggc agtacgacca gatccccgtg 360 gagatctgcg gccacaaggc catcggcacc gtgctggtgg gccccacccc cgtgaacatc 420 atcggccgca acctgctgac ccagatcggc tgcaccctga acttccccat cagccccatc 480 gagacggtgc ccgtgaagct gaagccgggg atggacggcc ccaaggtcaa gcagtggccc 540 ctgaccgagg agaagatcaa ggccctggtg gagatctgca ccgagatgga gaaggagggc 600 aagatcagca agatcggccc cgagaacccc tacaacaccc ccgtgttcgc catcaagaag 660 aaggacagca ccaagtggcg caagctggtg gacttccgcg agctgaacaa gcgcacccag 720 gacttctggg aggtgcagct gggcatcccc caccccgccg gcctgaagaa gaagaagagc 780 gtgaccgtgc tggacgtggg cgacgcctac ttcagcgtgc ccctggacaa ggacttccgc 840 aagtacaccg ccttcaccat ccccagcatc aacaacgaga cccccggcat ccgctaccag 900 tacaacgtgc tgccccaggg ctggaagggc agccccgcca tcttccagag cagcatgacc 960 aagatcctgg agcccttccg caagcagaac cccgacatcg tgatctacca gtacatggac 1020 gacctgtacg tgggcagcga cctggagatc ggccagcacc gcaccaagat cgaggagctg 1080 cgccagcacc tgctgcgctg gggcttcacc acccccgaca agaagcacca gaaggagccc 1140 cccttcctgt ggatgggcta cgagctgcac cccgacaagt ggaccgtgca gcccatcatg 1200 ctgcccgaga aggacagctg gaccgtgaac gacatccaga agctggtggg caagctgaac 1260 tgggccagcc agatctacgc cggcatcaag gtgaagcagc tgtgcaagct gctgcgcggc 1320 accaaggccc tgaccgaggt gatccccctg accgaggagg ccgagctgga gctggccgag 1380 aaccgcgaga tcctgaagga gcccgtgcac gaggtgtact acgaccccag caaggacctg 1440 gtggccgaga tccagaagca gggccagggc cagtggacct accagatcta ccaggagccc 1500 ttcaagaacc tgaagaccgg caagtacgcc cgcatgcgcg gcgcccacac caacgacgtg 1560 aagcagctga ccgaggccgt gcagaaggtg agcaccgaga gcatcgtgat ctggggcaag 1620 atccccaagt tcaagctgcc catccagaag gagacctggg aggcctggtg gatggagtac 1680 tggcaggcca cctggatccc cgagtgggag ttcgtgaaca ccccccccct ggtgaagctg 1740 tggtaccagc tggagaagga gcccatcgtg ggcgccgaga ccttctacgt ggacggcgcc 1800 gccaaccgcg agaccaagct gggcaaggcc ggctacgtga ccgaccgggg ccggcagaag 1860 gtggtgagca tcgccgacac caccaaccag aagaccgagc tgcaggccat ccacctggcc 1920 ctgcaggaca gcggcctgga ggtgaacatc gtgaccgaca gccagtacgc cctgggcatc 1980 atccaggccc agcccgacaa gagcgagagc gagctggtga gccagatcat cgagcagctg 2040 atcaagaagg agaaggtgta cctggcctgg gtgcccgccc acaagggcat cggcggcaac 2100 gagcaggtgg acaagctggt gagcgccggc atccgcaagg tgctg 2145 36 297 DNA Artificial Sequence Description of Artificial Sequence prot.opt.SF2 36 ccccagatca ccctgtggca gcgccccctg gtgaccatca ggatcggcgg ccagctcaag 60 gaggcgctgc tcgacaccgg cgccgacgac accgtgctgg aggagatgaa cctgcccggc 120 aagtggaagc ccaagatgat cggcgggatc gggggcttca tcaaggtgcg gcagtacgac 180 cagatccccg tggagatctg cggccacaag gccatcggca ccgtgctggt gggccccacc 240 cccgtgaaca tcatcggccg caacctgctg acccagatcg gctgcaccct gaacttc 297 37 297 DNA Artificial Sequence Description of Artificial Sequence protIna.opt.SF2 37 ccccagatca ccctgtggca gcgccccctg gtgaccatca ggatcggcgg ccagctcaag 60 gaggcgctgc tcgccaccgg cgccgacgac accgtgctgg aggagatgaa cctgcccggc 120 aagtggaagc ccaagatgat cggcgggatc gggggcttca tcaaggtgcg gcagtacgac 180 cagatccccg tggagatctg cggccacaag gccatcggca ccgtgctggt gggccccacc 240 cccgtgaaca tcatcggccg caacctgctg acccagatcg gctgcaccct gaacttc 297 38 1971 DNA Artificial Sequence Description of Artificial Sequence protInaRT.YM.opt.SF2 38 ccccagatca ccctgtggca gcgccccctg gtgaccatca ggatcggcgg ccagctcaag 60 gaggcgctgc tcgccaccgg cgccgacgac accgtgctgg aggagatgaa cctgcccggc 120 aagtggaagc ccaagatgat cggcgggatc gggggcttca tcaaggtgcg gcagtacgac 180 cagatccccg tggagatctg cggccacaag gccatcggca ccgtgctggt gggccccacc 240 cccgtgaaca tcatcggccg caacctgctg acccagatcg gctgcaccct gaacttcccc 300 atcagcccca tcgagacggt gcccgtgaag ctgaagccgg ggatggacgg ccccaaggtc 360 aagcagtggc ccctgaccga ggagaagatc aaggccctgg tggagatctg caccgagatg 420 gagaaggagg gcaagatcag caagatcggc cccgagaacc cctacaacac ccccgtgttc 480 gccatcaaga agaaggacag caccaagtgg cgcaagctgg tggacttccg cgagctgaac 540 aagcgcaccc aggacttctg ggaggtgcag ctgggcatcc cccaccccgc cggcctgaag 600 aagaagaaga gcgtgaccgt gctggacgtg ggcgacgcct acttcagcgt gcccctggac 660 aaggacttcc gcaagtacac cgccttcacc atccccagca tcaacaacga gacccccggc 720 atccgctacc agtacaacgt gctgccccag ggctggaagg gcagccccgc catcttccag 780 agcagcatga ccaagatcct ggagcccttc cgcaagcaga accccgacat cgtgatctac 840 caggcccccc tgtacgtggg cagcgacctg gagatcggcc agcaccgcac caagatcgag 900 gagctgcgcc agcacctgct gcgctggggc ttcaccaccc ccgacaagaa gcaccagaag 960 gagcccccct tcctgtggat gggctacgag ctgcaccccg acaagtggac cgtgcagccc 1020 atcatgctgc ccgagaagga cagctggacc gtgaacgaca tccagaagct ggtgggcaag 1080 ctgaactggg ccagccagat ctacgccggc atcaaggtga agcagctgtg caagctgctg 1140 cgcggcacca aggccctgac cgaggtgatc cccctgaccg aggaggccga gctggagctg 1200 gccgagaacc gcgagatcct gaaggagccc gtgcacgagg tgtactacga ccccagcaag 1260 gacctggtgg ccgagatcca gaagcagggc cagggccagt ggacctacca gatctaccag 1320 gagcccttca agaacctgaa gaccggcaag tacgcccgca tgcgcggcgc ccacaccaac 1380 gacgtgaagc agctgaccga ggccgtgcag aaggtgagca ccgagagcat cgtgatctgg 1440 ggcaagatcc ccaagttcaa gctgcccatc cagaaggaga cctgggaggc ctggtggatg 1500 gagtactggc aggccacctg gatccccgag tgggagttcg tgaacacccc ccccctggtg 1560 aagctgtggt accagctgga gaaggagccc atcgtgggcg ccgagacctt ctacgtggac 1620 ggcgccgcca accgcgagac caagctgggc aaggccggct acgtgaccga ccggggccgg 1680 cagaaggtgg tgagcatcgc cgacaccacc aaccagaaga ccgagctgca ggccatccac 1740 ctggccctgc aggacagcgg cctggaggtg aacatcgtga ccgacagcca gtacgccctg 1800 ggcatcatcc aggcccagcc cgacaagagc gagagcgagc tggtgagcca gatcatcgag 1860 cagctgatca agaaggagaa ggtgtacctg gcctgggtgc ccgcccacaa gggcatcggc 1920 ggcaacgagc aggtggacaa gctggtgagc gccggcatcc gcaaggtgct g 1971 39 2262 DNA Artificial Sequence Description of Artificial Sequence protInaRT.YMWM.opt.SF2 39 ccccagatca ccctgtggca gcgccccctg gtgaccatca ggatcggcgg ccagctcaag 60 gaggcgctgc tcgccaccgg cgccgacgac accgtgctgg aggagatgaa cctgcccggc 120 aagtggaagc ccaagatgat cggcgggatc gggggcttca tcaaggtgcg gcagtacgac 180 cagatccccg tggagatctg cggccacaag gccatcggca ccgtgctggt gggccccacc 240 cccgtgaaca tcatcggccg caacctgctg acccagatcg gctgcaccct gaacttcccc 300 cagatcaccc tgtggcagcg ccccctggtg accatcagga tcggcggcca gctcaaggag 360 gcgctgctcg acaccggcgc cgacgacacc gtgctggagg agatgaacct gcccggcaag 420 tggaagccca agatgatcgg cgggatcggg ggcttcatca aggtgcggca gtacgaccag 480 atccccgtgg agatctgcgg ccacaaggcc atcggcaccg tgctggtggg ccccaccccc 540 gtgaacatca tcggccgcaa cctgctgacc cagatcggct gcaccctgaa cttccccatc 600 agccccatcg agacggtgcc cgtgaagctg aagccgggga tggacggccc caaggtcaag 660 cagtggcccc tgaccgagga gaagatcaag gccctggtgg agatctgcac cgagatggag 720 aaggagggca agatcagcaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 780 atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 840 cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 900 aagaagagcg tgaccgtgct ggacgtgggc gacgcctact tcagcgtgcc cctggacaag 960 gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 1020 cgctaccagt acaacgtgct gccccagggc tggaagggca gccccgccat cttccagagc 1080 agcatgacca agatcctgga gcccttccgc aagcagaacc ccgacatcgt gatctaccag 1140 gcccccctgt acgtgggcag cgacctggag atcggccagc accgcaccaa gatcgaggag 1200 ctgcgccagc acctgctgcg ctggggcttc accacccccg acaagaagca ccagaaggag 1260 ccccccttcc tgcccatcga gctgcacccc gacaagtgga ccgtgcagcc catcatgctg 1320 cccgagaagg acagctggac cgtgaacgac atccagaagc tggtgggcaa gctgaactgg 1380 gccagccaga tctacgccgg catcaaggtg aagcagctgt gcaagctgct gcgcggcacc 1440 aaggccctga ccgaggtgat ccccctgacc gaggaggccg agctggagct ggccgagaac 1500 cgcgagatcc tgaaggagcc cgtgcacgag gtgtactacg accccagcaa ggacctggtg 1560 gccgagatcc agaagcaggg ccagggccag tggacctacc agatctacca ggagcccttc 1620 aagaacctga agaccggcaa gtacgcccgc atgcgcggcg cccacaccaa cgacgtgaag 1680 cagctgaccg aggccgtgca gaaggtgagc accgagagca tcgtgatctg gggcaagatc 1740 cccaagttca agctgcccat ccagaaggag acctgggagg cctggtggat ggagtactgg 1800 caggccacct ggatccccga gtgggagttc gtgaacaccc cccccctggt gaagctgtgg 1860 taccagctgg agaaggagcc catcgtgggc gccgagacct tctacgtgga cggcgccgcc 1920 aaccgcgaga ccaagctggg caaggccggc tacgtgaccg accggggccg gcagaaggtg 1980 gtgagcatcg ccgacaccac caaccagaag accgagctgc aggccatcca cctggccctg 2040 caggacagcg gcctggaggt gaacatcgtg accgacagcc agtacgccct gggcatcatc 2100 caggcccagc ccgacaagag cgagagcgag ctggtgagcc agatcatcga gcagctgatc 2160 aagaaggaga aggtgtacct ggcctgggtg cccgcccaca agggcatcgg cggcaacgag 2220 caggtggaca agctggtgag cgccggcatc cgcaaggtgc tg 2262 40 1990 DNA Artificial Sequence Description of Artificial Sequence ProtInaRTmut.SF2 40 gtcgacgcca ccatgcccca gatcaccctg tggcagcgcc ccctggtgac catcaggatc 60 ggcggccagc tcaaggaggc gctgctcgcc accggcgccg acgacaccgt gctggaggag 120 atgaacctgc ccggcaagtg gaagcccaag atgatcggcg ggatcggggg cttcatcaag 180 gtgcggcagt acgaccagat ccccgtggag atctgcggcc acaaggccat cggcaccgtg 240 ctggtgggcc ccacccccgt gaacatcatc ggccgcaacc tgctgaccca gatcggctgc 300 accctgaact tccccatcag ccccatcgag acggtgcccg tgaagctgaa gccggggatg 360 gacggcccca aggtcaagca gtggcccctg accgaggaga agatcaaggc cctggtggag 420 atctgcaccg agatggagaa ggagggcaag atcagcaaga tcggccccga gaacccctac 480 aacacccccg tgttcgccat caagaagaag gacagcacca agtggcgcaa gctggtggac 540 ttccgcgagc tgaacaagcg cacccaggac ttctgggagg tgcagctggg catcccccac 600 cccgccggcc tgaagaagaa gaagagcgtg accgtgctgg acgtgggcga cgcctacttc 660 agcgtgcccc tggacaagga cttccgcaag tacaccgcct tcaccatccc cagcatcaac 720 aacgagaccc ccggcatccg ctaccagtac aacgtgctgc cccagggctg gaagggcagc 780 cccgccatct tccagagcag catgaccaag atcctggagc ccttccgcaa gcagaacccc 840 gacatcgtga tctaccaggc ccccctgtac gtgggcagcg acctggagat cggccagcac 900 cgcaccaaga tcgaggagct gcgccagcac ctgctgcgct ggggcttcac cacccccgac 960 aagaagcacc agaaggagcc ccccttcctg cccatcgagc tgcaccccga caagtggacc 1020 gtgcagccca tcatgctgcc cgagaaggac agctggaccg tgaacgacat ccagaagctg 1080 gtgggcaagc tgaactgggc cagccagatc tacgccggca tcaaggtgaa gcagctgtgc 1140 aagctgctgc gcggcaccaa ggccctgacc gaggtgatcc ccctgaccga ggaggccgag 1200 ctggagctgg ccgagaaccg cgagatcctg aaggagcccg tgcacgaggt gtactacgac 1260 cccagcaagg acctggtggc cgagatccag aagcagggcc agggccagtg gacctaccag 1320 atctaccagg agcccttcaa gaacctgaag accggcaagt acgcccgcat gcgcggcgcc 1380 cacaccaacg acgtgaagca gctgaccgag gccgtgcaga aggtgagcac cgagagcatc 1440 gtgatctggg gcaagatccc caagttcaag ctgcccatcc agaaggagac ctgggaggcc 1500 tggtggatgg agtactggca ggccacctgg atccccgagt gggagttcgt gaacaccccc 1560 cccctggtga agctgtggta ccagctggag aaggagccca tcgtgggcgc cgagaccttc 1620 tacgtggacg gcgccgccaa ccgcgagacc aagctgggca aggccggcta cgtgaccgac 1680 cggggccggc agaaggtggt gagcatcgcc gacaccacca accagaagac cgagctgcag 1740 gccatccacc tggccctgca ggacagcggc ctggaggtga acatcgtgac cgacagccag 1800 tacgccctgg gcatcatcca ggcccagccc gacaagagcg agagcgagct ggtgagccag 1860 atcatcgagc agctgatcaa gaaggagaag gtgtacctgg cctgggtgcc cgcccacaag 1920 ggcatcggcg gcaacgagca ggtggacaag ctggtgagcg ccggcatccg caaggtgctc 1980 taaatctaga 1990 41 1977 DNA Artificial Sequence Description of Artificial Sequence protRT.opt.SF2 41 ccccagatca ccctgtggca gcgccccctg gtgaccatca ggatcggcgg ccagctcaag 60 gaggcgctgc tcgacaccgg cgccgacgac accgtgctgg aggagatgaa cctgcccggc 120 aagtggaagc ccaagatgat cggcgggatc gggggcttca tcaaggtgcg gcagtacgac 180 cagatccccg tggagatctg cggccacaag gccatcggca ccgtgctggt gggccccacc 240 cccgtgaaca tcatcggccg caacctgctg acccagatcg gctgcaccct gaacttcccc 300 atcagcccca tcgagacggt gcccgtgaag ctgaagccgg ggatggacgg ccccaaggtc 360 aagcagtggc ccctgaccga ggagaagatc aaggccctgg tggagatctg caccgagatg 420 gagaaggagg gcaagatcag caagatcggc cccgagaacc cctacaacac ccccgtgttc 480 gccatcaaga agaaggacag caccaagtgg cgcaagctgg tggacttccg cgagctgaac 540 aagcgcaccc aggacttctg ggaggtgcag ctgggcatcc cccaccccgc cggcctgaag 600 aagaagaaga gcgtgaccgt gctggacgtg ggcgacgcct acttcagcgt gcccctggac 660 aaggacttcc gcaagtacac cgccttcacc atccccagca tcaacaacga gacccccggc 720 atccgctacc agtacaacgt gctgccccag ggctggaagg gcagccccgc catcttccag 780 agcagcatga ccaagatcct ggagcccttc cgcaagcaga accccgacat cgtgatctac 840 cagtacatgg acgacctgta cgtgggcagc gacctggaga tcggccagca ccgcaccaag 900 atcgaggagc tgcgccagca cctgctgcgc tggggcttca ccacccccga caagaagcac 960 cagaaggagc cccccttcct gtggatgggc tacgagctgc accccgacaa gtggaccgtg 1020 cagcccatca tgctgcccga gaaggacagc tggaccgtga acgacatcca gaagctggtg 1080 ggcaagctga actgggccag ccagatctac gccggcatca aggtgaagca gctgtgcaag 1140 ctgctgcgcg gcaccaaggc cctgaccgag gtgatccccc tgaccgagga ggccgagctg 1200 gagctggccg agaaccgcga gatcctgaag gagcccgtgc acgaggtgta ctacgacccc 1260 agcaaggacc tggtggccga gatccagaag cagggccagg gccagtggac ctaccagatc 1320 taccaggagc ccttcaagaa cctgaagacc ggcaagtacg cccgcatgcg cggcgcccac 1380 accaacgacg tgaagcagct gaccgaggcc gtgcagaagg tgagcaccga gagcatcgtg 1440 atctggggca agatccccaa gttcaagctg cccatccaga aggagacctg ggaggcctgg 1500 tggatggagt actggcaggc cacctggatc cccgagtggg agttcgtgaa cacccccccc 1560 ctggtgaagc tgtggtacca gctggagaag gagcccatcg tgggcgccga gaccttctac 1620 gtggacggcg ccgccaaccg cgagaccaag ctgggcaagg ccggctacgt gaccgaccgg 1680 ggccggcaga aggtggtgag catcgccgac accaccaacc agaagaccga gctgcaggcc 1740 atccacctgg ccctgcagga cagcggcctg gaggtgaaca tcgtgaccga cagccagtac 1800 gccctgggca tcatccaggc ccagcccgac aagagcgaga gcgagctggt gagccagatc 1860 atcgagcagc tgatcaagaa ggagaaggtg tacctggcct gggtgcccgc ccacaagggc 1920 atcggcggca acgagcaggt ggacaagctg gtgagcgccg gcatccgcaa ggtgctg 1977 42 3252 DNA Artificial Sequence Description of Artificial Sequence ProtRT.TatRevNef.opt_B 42 tgccccaga tcaccctgtg gcagcgcccc ctggtgacca tcaggatcgg cggccagctc 60 aaggaggcgc tgctcgccac cggcgccgac gacaccgtgc tggaggagat gaacctgccc 120 ggcaagtgga agcccaagat gatcggcggg atcgggggct tcatcaaggt gcggcagtac 180 gaccagatcc ccgtggagat ctgcggccac aaggccatcg gcaccgtgct ggtgggcccc 240 acccccgtga acatcatcgg ccgcaacctg ctgacccaga tcggctgcac cctgaacttc 300 cccatcagcc ccatcgagac ggtgcccgtg aagctgaagc cggggatgga cggccccaag 360 gtcaagcagt ggcccctgac cgaggagaag atcaaggccc tggtggagat ctgcaccgag 420 atggagaagg agggcaagat cagcaagatc ggccccgaga acccctacaa cacccccgtg 480 ttcgccatca agaagaagga cagcaccaag tggcgcaagc tggtggactt ccgcgagctg 540 aacaagcgca cccaggactt ctgggaggtg cagctgggca tcccccaccc cgccggcctg 600 aagaagaaga agagcgtgac cgtgctggac gtgggcgacg cctacttcag cgtgcccctg 660 gacaaggact tccgcaagta caccgccttc accatcccca gcatcaacaa cgagaccccc 720 ggcatccgct accagtacaa cgtgctgccc cagggctgga agggcagccc cgccatcttc 780 cagagcagca tgaccaagat cctggagccc ttccgcaagc agaaccccga catcgtgatc 840 taccaggccc ccctgtacgt gggcagcgac ctggagatcg gccagcaccg caccaagatc 900 gaggagctgc gccagcacct gctgcgctgg ggcttcacca cccccgacaa gaagcaccag 960 aaggagcccc ccttcctgcc catcgagctg caccccgaca agtggaccgt gcagcccatc 1020 atgctgcccg agaaggacag ctggaccgtg aacgacatcc agaagctggt gggcaagctg 1080 aactgggcca gccagatcta cgccggcatc aaggtgaagc agctgtgcaa gctgctgcgc 1140 ggcaccaagg ccctgaccga ggtgatcccc ctgaccgagg aggccgagct ggagctggcc 1200 gagaaccgcg agatcctgaa ggagcccgtg cacgaggtgt actacgaccc cagcaaggac 1260 ctggtggccg agatccagaa gcagggccag ggccagtgga cctaccagat ctaccaggag 1320 cccttcaaga acctgaagac cggcaagtac gcccgcatgc gcggcgccca caccaacgac 1380 gtgaagcagc tgaccgaggc cgtgcagaag gtgagcaccg agagcatcgt gatctggggc 1440 aagatcccca agttcaagct gcccatccag aaggagacct gggaggcctg gtggatggag 1500 tactggcagg ccacctggat ccccgagtgg gagttcgtga acaccccccc cctggtgaag 1560 ctgtggtacc agctggagaa ggagcccatc gtgggcgccg agaccttcta cgtggacggc 1620 gccgccaacc gcgagaccaa gctgggcaag gccggctacg tgaccgaccg gggccggcag 1680 aaggtggtga gcatcgccga caccaccaac cagaagaccg agctgcaggc catccacctg 1740 gccctgcagg acagcggcct ggaggtgaac atcgtgaccg acagccagta cgccctgggc 1800 atcatccagg cccagcccga caagagcgag agcgagctgg tgagccagat catcgagcag 1860 ctgatcaaga aggagaaggt gtacctggcc tgggtgcccg cccacaaggg catcggcggc 1920 aacgagcagg tggacaagct ggtgagcgcc ggcatccgca aggtgctcga attcgagccc 1980 gtggaccccc gcctggagcc ctggaagcac cccggcagcc agcccaagac cgccggcacc 2040 aactgctact gcaagaagtg ctgcttccac tgccaggtga gcttcatcac caagggcctg 2100 ggcatcagct acggccgcaa gaagcgccgc cagcgccgcc gcgccccccc cgacagcgag

2160 gtgcaccagg tgagcctgcc caagcagccc gccagccagc cccagggcga ccccaccggc 2220 cccaaggaga gcaagaagaa ggtggagcgc gagaccgaga ccgaccccgt gcaccccggg 2280 gccggccgca gcggcgacag cgacgaggag ctgctgcaga ccgtgcgctt catcaagttc 2340 ctgtaccaga gcaaccccct gcccagcccc aagggcaccc gccaggccga cctgaaccgc 2400 cgccgccgct ggcgcgagcg ccagcgccag atccagagca tcagcgcctg gatcatcagc 2460 acccacctgg gccgcagcac cgagcccgtg cccctgcagc tgccccccga cctgcgcctg 2520 aacctggact gcagcgagga ctgcggcacc agcggcaccc agggcgtggg cagcccccag 2580 gtgctgggcg agagccccgc cgtgctggac agcggcacca aggagctcga ggccggcaag 2640 tggagcaagc gcatgagcgg ctggagcgcc gtgcgcgagc gcatgaagcg cgccgagccc 2700 gccgagcccg ccgccgacgg cgtgggcgcc gtgagccgcg acctggagaa gcacggcgcc 2760 atcaccagca gcaacaccgc cgccaacaac gccgactgcg cctggctgga ggcccaggag 2820 gacgaggacg tgggcttccc cgtgcgcccc caggtgcccc tgcgccccat gacctacaag 2880 gccgccctgg acctgagcca cttcctgaag gagaagggcg gcctggaggg cctgatctac 2940 agccagaagc gccaggacat cctggacctg tggatccacc acacccaggg ctacttcccc 3000 ggctggcaga actacacccc cggccccggc atccgctacc ccctgacctt cggctggtgc 3060 ttcaagctgg tgcccgtgga ccccgactac gtggaggagg ccaacgccgg cgagaacaac 3120 agcctgctgc accccatgag ccagcacggc atggacgacc ccgagaagga ggtgctggtg 3180 tggcgcttcg acagccgcct ggccttccac cacatggccc gcgagctgca ccccgagtac 3240 tacaaggact gc 3252 43 3264 DNA Artificial Sequence Description of Artificial Sequence ProtRTTatRevNef.opt_B 43 gccaccatgc cccagatcac cctgtggcag cgccccctgg tgaccatcag gatcggcggc 60 cagctcaagg aggcgctgct cgccaccggc gccgacgaca ccgtgctgga ggagatgaac 120 ctgcccggca agtggaagcc caagatgatc ggcgggatcg ggggcttcat caaggtgcgg 180 cagtacgacc agatccccgt ggagatctgc ggccacaagg ccatcggcac cgtgctggtg 240 ggccccaccc ccgtgaacat catcggccgc aacctgctga cccagatcgg ctgcaccctg 300 aacttcccca tcagccccat cgagacggtg cccgtgaagc tgaagccggg gatggacggc 360 cccaaggtca agcagtggcc cctgaccgag gagaagatca aggccctggt ggagatctgc 420 accgagatgg agaaggaggg caagatcagc aagatcggcc ccgagaaccc ctacaacacc 480 cccgtgttcg ccatcaagaa gaaggacagc accaagtggc gcaagctggt ggacttccgc 540 gagctgaaca agcgcaccca ggacttctgg gaggtgcagc tgggcatccc ccaccccgcc 600 ggcctgaaga agaagaagag cgtgaccgtg ctggacgtgg gcgacgccta cttcagcgtg 660 cccctggaca aggacttccg caagtacacc gccttcacca tccccagcat caacaacgag 720 acccccggca tccgctacca gtacaacgtg ctgccccagg gctggaaggg cagccccgcc 780 atcttccaga gcagcatgac caagatcctg gagcccttcc gcaagcagaa ccccgacatc 840 gtgatctacc aggcccccct gtacgtgggc agcgacctgg agatcggcca gcaccgcacc 900 aagatcgagg agctgcgcca gcacctgctg cgctggggct tcaccacccc cgacaagaag 960 caccagaagg agcccccctt cctgcccatc gagctgcacc ccgacaagtg gaccgtgcag 1020 cccatcatgc tgcccgagaa ggacagctgg accgtgaacg acatccagaa gctggtgggc 1080 aagctgaact gggccagcca gatctacgcc ggcatcaagg tgaagcagct gtgcaagctg 1140 ctgcgcggca ccaaggccct gaccgaggtg atccccctga ccgaggaggc cgagctggag 1200 ctggccgaga accgcgagat cctgaaggag cccgtgcacg aggtgtacta cgaccccagc 1260 aaggacctgg tggccgagat ccagaagcag ggccagggcc agtggaccta ccagatctac 1320 caggagccct tcaagaacct gaagaccggc aagtacgccc gcatgcgcgg cgcccacacc 1380 aacgacgtga agcagctgac cgaggccgtg cagaaggtga gcaccgagag catcgtgatc 1440 tggggcaaga tccccaagtt caagctgccc atccagaagg agacctggga ggcctggtgg 1500 atggagtact ggcaggccac ctggatcccc gagtgggagt tcgtgaacac cccccccctg 1560 gtgaagctgt ggtaccagct ggagaaggag cccatcgtgg gcgccgagac cttctacgtg 1620 gacggcgccg ccaaccgcga gaccaagctg ggcaaggccg gctacgtgac cgaccggggc 1680 cggcagaagg tggtgagcat cgccgacacc accaaccaga agaccgagct gcaggccatc 1740 cacctggccc tgcaggacag cggcctggag gtgaacatcg tgaccgacag ccagtacgcc 1800 ctgggcatca tccaggccca gcccgacaag agcgagagcg agctggtgag ccagatcatc 1860 gagcagctga tcaagaagga gaaggtgtac ctggcctggg tgcccgccca caagggcatc 1920 ggcggcaacg agcaggtgga caagctggtg agcgccggca tccgcaaggt gctcgaattc 1980 gagcccgtgg acccccgcct ggagccctgg aagcaccccg gcagccagcc caagaccgcc 2040 ggcaccaact gctactgcaa gaagtgctgc ttccactgcc aggtgagctt catcaccaag 2100 ggcctgggca tcagctacgg ccgcaagaag cgccgccagc gccgccgcgc cccccccgac 2160 agcgaggtgc accaggtgag cctgcccaag cagcccgcca gccagcccca gggcgacccc 2220 accggcccca aggagagcaa gaagaaggtg gagcgcgaga ccgagaccga ccccgtgcac 2280 cccggggccg gccgcagcgg cgacagcgac gaggagctgc tgcagaccgt gcgcttcatc 2340 aagttcctgt accagagcaa ccccctgccc agccccaagg gcacccgcca ggccgacctg 2400 aaccgccgcc gccgctggcg cgagcgccag cgccagatcc agagcatcag cgcctggatc 2460 atcagcaccc acctgggccg cagcaccgag cccgtgcccc tgcagctgcc ccccgacctg 2520 cgcctgaacc tggactgcag cgaggactgc ggcaccagcg gcacccaggg cgtgggcagc 2580 ccccaggtgc tgggcgagag ccccgccgtg ctggacagcg gcaccaagga gctcgaggcc 2640 ggcaagtgga gcaagcgcat gagcggctgg agcgccgtgc gcgagcgcat gaagcgcgcc 2700 gagcccgccg agcccgccgc cgacggcgtg ggcgccgtga gccgcgacct ggagaagcac 2760 ggcgccatca ccagcagcaa caccgccgcc aacaacgccg actgcgcctg gctggaggcc 2820 caggaggacg aggacgtggg cttccccgtg cgcccccagg tgcccctgcg ccccatgacc 2880 tacaaggccg ccctggacct gagccacttc ctgaaggaga agggcggcct ggagggcctg 2940 atctacagcc agaagcgcca ggacatcctg gacctgtgga tccaccacac ccagggctac 3000 ttccccggct ggcagaacta cacccccggc cccggcatcc gctaccccct gaccttcggc 3060 tggtgcttca agctggtgcc cgtggacccc gactacgtgg aggaggccaa cgccggcgag 3120 aacaacagcc tgctgcaccc catgagccag cacggcatgg acgaccccga gaaggaggtg 3180 ctggtgtggc gcttcgacag ccgcctggcc ttccaccaca tggcccgcga gctgcacccc 3240 gagtactaca aggactgcga ttaa 3264 44 348 DNA Artificial Sequence Description of Artificial Sequence rev.exon1_2.M5-10.opt.SF162 44 atggccggcc gcagcggcga cagcgacgag gagctgctgc agaccgtgcg cttcatcaag 60 ttcctgtacc agagcaaccc cctgcccagc cccaagggca cccgccaggc cgacctgaac 120 cgccgccgcc gctggcgcga gcgccagcgc cagatccaga gcatcagcgc ctggatcatc 180 agcacccacc tgggccgcag caccgagccc gtgcccctgc agctgccccc cgacctgcgc 240 ctgaacctgg actgcagcga ggactgcggc accagcggca cccagggcgt gggcagcccc 300 caggtgctgg gcgagagccc cgccgtgctg gacagcggca ccaaggag 348 45 348 DNA Artificial Sequence Description of Artificial Sequence description 45 atggccggcc gcagcggcga cagcgacgag gagctgctgc agaccgtgcg cttcatcaag 60 ttcctgtacc agagcaaccc cctgcccagc cccaagggca cccgccaggc ccgccgcaac 120 cgccgccgcc gctggcgcga gcgccagcgc cagatccaga gcatcagcgc ctggatcatc 180 agcacccacc tgggccgcag caccgagccc gtgcccctgc agctgccccc cctggagcgc 240 ctgaacctgg actgcagcga ggactgcggc accagcggca cccagggcgt gggcagcccc 300 caggtgctgg gcgagagccc cgccgtgctg gacagcggca ccaaggag 348 46 1977 DNA Artificial Sequence Description of Artificial Sequence RT.opt.SF2 (mutant) 46 gccaccatgc cccagatcac cctgtggcag cgccccctgg tgaccatcag gatcggcggc 60 cagctcaagg aggcgctgct cgccaccggc gccgacgaca ccgtgctgga ggagatgaac 120 ctgcccggca agtggaagcc caagatgatc ggcgggatcg ggggcttcat caaggtgcgg 180 cagtacgacc agatccccgt ggagatctgc ggccacaagg ccatcggcac cgtgctggtg 240 ggccccaccc ccgtgaacat catcggccgc aacctgctga cccagatcgg ctgcaccctg 300 aacttcccca tcagccccat cgagacggtg cccgtgaagc tgaagccggg gatggacggc 360 cccaaggtca agcagtggcc cctgaccgag gagaagatca aggccctggt ggagatctgc 420 accgagatgg agaaggaggg caagatcagc aagatcggcc ccgagaaccc ctacaacacc 480 cccgtgttcg ccatcaagaa gaaggacagc accaagtggc gcaagctggt ggacttccgc 540 gagctgaaca agcgcaccca ggacttctgg gaggtgcagc tgggcatccc ccaccccgcc 600 ggcctgaaga agaagaagag cgtgaccgtg ctggacgtgg gcgacgccta cttcagcgtg 660 cccctggaca aggacttccg caagtacacc gccttcacca tccccagcat caacaacgag 720 acccccggca tccgctacca gtacaacgtg ctgccccagg gctggaaggg cagccccgcc 780 atcttccaga gcagcatgac caagatcctg gagcccttcc gcaagcagaa ccccgacatc 840 gtgatctacc aggcccccct gtacgtgggc agcgacctgg agatcggcca gcaccgcacc 900 aagatcgagg agctgcgcca gcacctgctg cgctggggct tcaccacccc cgacaagaag 960 caccagaagg agcccccctt cctgcccatc gagctgcacc ccgacaagtg gaccgtgcag 1020 cccatcatgc tgcccgagaa ggacagctgg accgtgaacg acatccagaa gctggtgggc 1080 aagctgaact gggccagcca gatctacgcc ggcatcaagg tgaagcagct gtgcaagctg 1140 ctgcgcggca ccaaggccct gaccgaggtg atccccctga ccgaggaggc cgagctggag 1200 ctggccgaga accgcgagat cctgaaggag cccgtgcacg aggtgtacta cgaccccagc 1260 aaggacctgg tggccgagat ccagaagcag ggccagggcc agtggaccta ccagatctac 1320 caggagccct tcaagaacct gaagaccggc aagtacgccc gcatgcgcgg cgcccacacc 1380 aacgacgtga agcagctgac cgaggccgtg cagaaggtga gcaccgagag catcgtgatc 1440 tggggcaaga tccccaagtt caagctgccc atccagaagg agacctggga ggcctggtgg 1500 atggagtact ggcaggccac ctggatcccc gagtgggagt tcgtgaacac cccccccctg 1560 gtgaagctgt ggtaccagct ggagaaggag cccatcgtgg gcgccgagac cttctacgtg 1620 gacggcgccg ccaaccgcga gaccaagctg ggcaaggccg gctacgtgac cgaccggggc 1680 cggcagaagg tggtgagcat cgccgacacc accaaccaga agaccgagct gcaggccatc 1740 cacctggccc tgcaggacag cggcctggag gtgaacatcg tgaccgacag ccagtacgcc 1800 ctgggcatca tccaggccca gcccgacaag agcgagagcg agctggtgag ccagatcatc 1860 gagcagctga tcaagaagga gaaggtgtac ctggcctggg tgcccgccca caagggcatc 1920 ggcggcaacg agcaggtgga caagctggtg agcgccggca tccgcaaggt gctctaa 1977 47 1989 DNA Artificial Sequence Description of Artificial Sequence description 47 gccaccatgc cccagatcac cctgtggcag cgccccctgg tgaccatcag gatcggcggc 60 cagctcaagg aggcgctgct cgacaccggc gccgacgaca ccgtgctgga ggagatgaac 120 ctgcccggca agtggaagcc caagatgatc ggcgggatcg ggggcttcat caaggtgcgg 180 cagtacgacc agatccccgt ggagatctgc ggccacaagg ccatcggcac cgtgctggtg 240 ggccccaccc ccgtgaacat catcggccgc aacctgctga cccagatcgg ctgcaccctg 300 aacttcccca tcagccccat cgagacggtg cccgtgaagc tgaagccggg gatggacggc 360 cccaaggtca agcagtggcc cctgaccgag gagaagatca aggccctggt ggagatctgc 420 accgagatgg agaaggaggg caagatcagc aagatcggcc ccgagaaccc ctacaacacc 480 cccgtgttcg ccatcaagaa gaaggacagc accaagtggc gcaagctggt ggacttccgc 540 gagctgaaca agcgcaccca ggacttctgg gaggtgcagc tgggcatccc ccaccccgcc 600 ggcctgaaga agaagaagag cgtgaccgtg ctggacgtgg gcgacgccta cttcagcgtg 660 cccctggaca aggacttccg caagtacacc gccttcacca tccccagcat caacaacgag 720 acccccggca tccgctacca gtacaacgtg ctgccccagg gctggaaggg cagccccgcc 780 atcttccaga gcagcatgac caagatcctg gagcccttcc gcaagcagaa ccccgacatc 840 gtgatctacc agtacatgga cgacctgtac gtgggcagcg acctggagat cggccagcac 900 cgcaccaaga tcgaggagct gcgccagcac ctgctgcgct ggggcttcac cacccccgac 960 aagaagcacc agaaggagcc ccccttcctg tggatgggct acgagctgca ccccgacaag 1020 tggaccgtgc agcccatcat gctgcccgag aaggacagct ggaccgtgaa cgacatccag 1080 aagctggtgg gcaagctgaa ctgggccagc cagatctacg ccggcatcaa ggtgaagcag 1140 ctgtgcaagc tgctgcgcgg caccaaggcc ctgaccgagg tgatccccct gaccgaggag 1200 gccgagctgg agctggccga gaaccgcgag atcctgaagg agcccgtgca cgaggtgtac 1260 tacgacccca gcaaggacct ggtggccgag atccagaagc agggccaggg ccagtggacc 1320 taccagatct accaggagcc cttcaagaac ctgaagaccg gcaagtacgc ccgcatgcgc 1380 ggcgcccaca ccaacgacgt gaagcagctg accgaggccg tgcagaaggt gagcaccgag 1440 agcatcgtga tctggggcaa gatccccaag ttcaagctgc ccatccagaa ggagacctgg 1500 gaggcctggt ggatggagta ctggcaggcc acctggatcc ccgagtggga gttcgtgaac 1560 accccccccc tggtgaagct gtggtaccag ctggagaagg agcccatcgt gggcgccgag 1620 accttctacg tggacggcgc cgccaaccgc gagaccaagc tgggcaaggc cggctacgtg 1680 accgaccggg gccggcagaa ggtggtgagc atcgccgaca ccaccaacca gaagaccgag 1740 ctgcaggcca tccacctggc cctgcaggac agcggcctgg aggtgaacat cgtgaccgac 1800 agccagtacg ccctgggcat catccaggcc cagcccgaca agagcgagag cgagctggtg 1860 agccagatca tcgagcagct gatcaagaag gagaaggtgt acctggcctg ggtgcccgcc 1920 cacaagggca tcggcggcaa cgagcaggtg gacaagctgg tgagcgccgg catccgcaag 1980 gtgctgtaa 1989 48 1693 DNA Artificial Sequence Description of Artificial Sequence RTmut.SF2 48 gtcgacgcca ccatgcccat cagccccatc gagacggtgc ccgtgaagct gaagccgggg 60 atggacggcc ccaaggtcaa gcagtggccc ctgaccgagg agaagatcaa ggccctggtg 120 gagatctgca ccgagatgga gaaggagggc aagatcagca agatcggccc cgagaacccc 180 tacaacaccc ccgtgttcgc catcaagaag aaggacagca ccaagtggcg caagctggtg 240 gacttccgcg agctgaacaa gcgcacccag gacttctggg aggtgcagct gggcatcccc 300 caccccgccg gcctgaagaa gaagaagagc gtgaccgtgc tggacgtggg cgacgcctac 360 ttcagcgtgc ccctggacaa ggacttccgc aagtacaccg ccttcaccat ccccagcatc 420 aacaacgaga cccccggcat ccgctaccag tacaacgtgc tgccccaggg ctggaagggc 480 agccccgcca tcttccagag cagcatgacc aagatcctgg agcccttccg caagcagaac 540 cccgacatcg tgatctacca ggcccccctg tacgtgggca gcgacctgga gatcggccag 600 caccgcacca agatcgagga gctgcgccag cacctgctgc gctggggctt caccaccccc 660 gacaagaagc accagaagga gccccccttc ctgcccatcg agctgcaccc cgacaagtgg 720 accgtgcagc ccatcatgct gcccgagaag gacagctgga ccgtgaacga catccagaag 780 ctggtgggca agctgaactg ggccagccag atctacgccg gcatcaaggt gaagcagctg 840 tgcaagctgc tgcgcggcac caaggccctg accgaggtga tccccctgac cgaggaggcc 900 gagctggagc tggccgagaa ccgcgagatc ctgaaggagc ccgtgcacga ggtgtactac 960 gaccccagca aggacctggt ggccgagatc cagaagcagg gccagggcca gtggacctac 1020 cagatctacc aggagccctt caagaacctg aagaccggca agtacgcccg catgcgcggc 1080 gcccacacca acgacgtgaa gcagctgacc gaggccgtgc agaaggtgag caccgagagc 1140 atcgtgatct ggggcaagat ccccaagttc aagctgccca tccagaagga gacctgggag 1200 gcctggtgga tggagtactg gcaggccacc tggatccccg agtgggagtt cgtgaacacc 1260 ccccccctgg tgaagctgtg gtaccagctg gagaaggagc ccatcgtggg cgccgagacc 1320 ttctacgtgg acggcgccgc caaccgcgag accaagctgg gcaaggccgg ctacgtgacc 1380 gaccggggcc ggcagaaggt ggtgagcatc gccgacacca ccaaccagaa gaccgagctg 1440 caggccatcc acctggccct gcaggacagc ggcctggagg tgaacatcgt gaccgacagc 1500 cagtacgccc tgggcatcat ccaggcccag cccgacaaga gcgagagcga gctggtgagc 1560 cagatcatcg agcagctgat caagaaggag aaggtgtacc tggcctgggt gcccgcccac 1620 aagggcatcg gcggcaacga gcaggtggac aagctggtga gcgccggcat ccgcaaggtg 1680 ctctaaagaa ttc 1693 49 303 DNA Artificial Sequence Description of Artificial Sequence tat.exon1_2.opt.C22-37.SF2 49 atggagcccg tggacccccg cctggagccc tggaagcacc ccggcagcca gcccaagacc 60 gccggcacca actgctactg caagaagtgc tgcttccact gccaggtgag cttcatcacc 120 aagggcctgg gcatcagcta cggccgcaag aagcgccgcc agcgccgccg cgcccccccc 180 gacagcgagg tgcaccaggt gagcctgccc aagcagcccg ccagccagcc ccagggcgac 240 cccaccggcc ccaaggagag caagaagaag gtggagcgcg agaccgagac cgaccccgtg 300 cac 303 50 303 DNA Artificial Sequence Description of Artificial Sequence description 50 atggagcccg tggacccccg cctggagccc tggaagcacc ccggcagcca gcccaagacc 60 gcctgcacca actgctactg caagaagtgc tgcttccact gccaggtgag cttcatcacc 120 aagggcctgg gcatcagcta cggccgcaag aagcgccgcc agcgccgccg cgcccccccc 180 gacagcgagg tgcaccaggt gagcctgccc aagcagcccg ccagccagcc ccagggcgac 240 cccaccggcc ccaaggagag caagaagaag gtggagcgcg agaccgagac cgaccccgtg 300 cac 303 51 1281 DNA Artificial Sequence Description of Artificial Sequence TatRevNef.opt.native.SF162 51 atggagcccg tggacccccg cctggagccc tggaagcacc ccggcagcca gcccaagacc 60 gcctgcacca actgctactg caagaagtgc tgcttccact gccaggtgtg cttcatcacc 120 aagggcctgg gcatcagcta cggccgcaag aagcgccgcc agcgccgccg cgcccccccc 180 gacagcgagg tgcaccaggt gagcctgccc aagcagcccg ccagccagcc ccagggcgac 240 cccaccggcc ccaaggagag caagaagaag gtggagcgcg agaccgagac cgaccccgtg 300 caccccgggg ccggccgcag cggcgacagc gacgaggagc tgctgcagac cgtgcgcttc 360 atcaagttcc tgtaccagag caaccccctg cccagcccca agggcacccg ccaggcccgc 420 cgcaaccgcc gccgccgctg gcgcgagcgc cagcgccaga tccagagcat cagcgcctgg 480 atcatcagca cccacctggg ccgcagcacc gagcccgtgc ccctgcagct gccccccctg 540 gagcgcctga acctggactg cagcgaggac tgcggcacca gcggcaccca gggcgtgggc 600 agcccccagg tgctgggcga gagccccgcc gtgctggaca gcggcaccaa ggagctcgag 660 ggcggcaagt ggagcaagcg catgagcggc tggagcgccg tgcgcgagcg catgaagcgc 720 gccgagcccg ccgagcccgc cgccgacggc gtgggcgccg tgagccgcga cctggagaag 780 cacggcgcca tcaccagcag caacaccgcc gccaacaacg ccgactgcgc ctggctggag 840 gcccaggagg acgaggacgt gggcttcccc gtgcgccccc aggtgcccct gcgccccatg 900 acctacaagg ccgccctgga cctgagccac ttcctgaagg agaagggcgg cctggagggc 960 ctgatctaca gccagaagcg ccaggacatc ctggacctgt ggatccacca cacccagggc 1020 tacttccccg actggcagaa ctacaccccc ggccccggca tccgctaccc cctgaccttc 1080 ggctggtgct tcaagctggt gcccgtggac cccgactacg tggaggaggc caacgccggc 1140 gagaacaaca gcctgctgca ccccatgagc cagcacggca tggacgaccc cgagaaggag 1200 gtgctggtgt ggcgcttcga cagccgcctg gccttccacc acatggcccg cgagctgcac 1260 cccgagtact acaaggactg c 1281 52 1281 DNA Artificial Sequence Description of Artificial Sequence TatRevNef.opt.SF162 52 atggagcccg tggacccccg cctggagccc tggaagcacc ccggcagcca gcccaagacc 60 gccggcacca actgctactg caagaagtgc tgcttccact gccaggtgag cttcatcacc 120 aagggcctgg gcatcagcta cggccgcaag aagcgccgcc agcgccgccg cgcccccccc 180 gacagcgagg tgcaccaggt gagcctgccc aagcagcccg ccagccagcc ccagggcgac 240 cccaccggcc ccaaggagag caagaagaag gtggagcgcg agaccgagac cgaccccgtg 300 caccccgggg ccggccgcag cggcgacagc gacgaggagc tgctgcagac cgtgcgcttc 360 atcaagttcc tgtaccagag caaccccctg cccagcccca agggcacccg ccaggccgac 420 ctgaaccgcc gccgccgctg gcgcgagcgc cagcgccaga tccagagcat cagcgcctgg 480 atcatcagca cccacctggg ccgcagcacc gagcccgtgc ccctgcagct gccccccgac 540 ctgcgcctga acctggactg cagcgaggac tgcggcacca gcggcaccca gggcgtgggc 600 agcccccagg tgctgggcga gagccccgcc gtgctggaca gcggcaccaa ggagctcgag 660 gccggcaagt ggagcaagcg catgagcggc tggagcgccg tgcgcgagcg catgaagcgc 720 gccgagcccg ccgagcccgc cgccgacggc gtgggcgccg tgagccgcga cctggagaag 780 cacggcgcca tcaccagcag caacaccgcc gccaacaacg ccgactgcgc ctggctggag 840 gcccaggagg acgaggacgt gggcttcccc gtgcgccccc aggtgcccct gcgccccatg 900 acctacaagg ccgccctgga cctgagccac ttcctgaagg agaagggcgg cctggagggc 960 ctgatctaca gccagaagcg ccaggacatc ctggacctgt ggatccacca cacccagggc 1020 tacttccccg gctggcagaa ctacaccccc ggccccggca tccgctaccc cctgaccttc 1080 ggctggtgct tcaagctggt gcccgtggac cccgactacg tggaggaggc caacgccggc 1140 gagaacaaca gcctgctgca ccccatgagc cagcacggca tggacgaccc cgagaaggag 1200 gtgctggtgt ggcgcttcga cagccgcctg gccttccacc acatggcccg cgagctgcac 1260

cccgagtact acaaggactg c 1281 53 2799 DNA Artificial Sequence Description of Artificial Sequence TatRevNefGag B 53 gccaccatgg agcccgtgga cccccgcctg gagccctgga agcaccccgg cagccagccc 60 aagaccgccg gcaccaactg ctactgcaag aagtgctgct tccactgcca ggtgagcttc 120 atcaccaagg gcctgggcat cagctacggc cgcaagaagc gccgccagcg ccgccgcgcc 180 ccccccgaca gcgaggtgca ccaggtgagc ctgcccaagc agcccgccag ccagccccag 240 ggcgacccca ccggccccaa ggagagcaag aagaaggtgg agcgcgagac cgagaccgac 300 cccgtgcacc ccggggccgg ccgcagcggc gacagcgacg aggagctgct gcagaccgtg 360 cgcttcatca agttcctgta ccagagcaac cccctgccca gccccaaggg cacccgccag 420 gccgacctga accgccgccg ccgctggcgc gagcgccagc gccagatcca gagcatcagc 480 gcctggatca tcagcaccca cctgggccgc agcaccgagc ccgtgcccct gcagctgccc 540 cccgacctgc gcctgaacct ggactgcagc gaggactgcg gcaccagcgg cacccagggc 600 gtgggcagcc cccaggtgct gggcgagagc cccgccgtgc tggacagcgg caccaaggag 660 ctcgaggccg gcaagtggag caagcgcatg agcggctgga gcgccgtgcg cgagcgcatg 720 aagcgcgccg agcccgccga gcccgccgcc gacggcgtgg gcgccgtgag ccgcgacctg 780 gagaagcacg gcgccatcac cagcagcaac accgccgcca acaacgccga ctgcgcctgg 840 ctggaggccc aggaggacga ggacgtgggc ttccccgtgc gcccccaggt gcccctgcgc 900 cccatgacct acaaggccgc cctggacctg agccacttcc tgaaggagaa gggcggcctg 960 gagggcctga tctacagcca gaagcgccag gacatcctgg acctgtggat ccaccacacc 1020 cagggctact tccccggctg gcagaactac acccccggcc ccggcatccg ctaccccctg 1080 accttcggct ggtgcttcaa gctggtgccc gtggaccccg actacgtgga ggaggccaac 1140 gccggcgaga acaacagcct gctgcacccc atgagccagc acggcatgga cgaccccgag 1200 aaggaggtgc tggtgtggcg cttcgacagc cgcctggcct tccaccacat ggcccgcgag 1260 ctgcaccccg agtactacaa ggactgcgaa ttcggcgccc gcgccagcgt gctgagcggc 1320 ggcgagctgg acaagtggga gaagatccgc ctgcgccccg gcggcaagaa gaagtacaag 1380 ctgaagcaca tcgtgtgggc cagccgcgag ctggagcgct tcgccgtgaa ccccggcctg 1440 ctggagacca gcgagggctg ccgccagatc ctgggccagc tgcagcccag cctgcagacc 1500 ggcagcgagg agctgcgcag cctgtacaac accgtggcca ccctgtactg cgtgcaccag 1560 cgcatcgacg tcaaggacac caaggaggcc ctggagaaga tcgaggagga gcagaacaag 1620 tccaagaaga aggcccagca ggccgccgcc gccgccggca ccggcaacag cagccaggtg 1680 agccagaact accccatcgt gcagaacctg cagggccaga tggtgcacca ggccatcagc 1740 ccccgcaccc tgaacgcctg ggtgaaggtg gtggaggaga aggccttcag ccccgaggtg 1800 atccccatgt tcagcgccct gagcgagggc gccacccccc aggacctgaa cacgatgttg 1860 aacaccgtgg gcggccacca ggccgccatg cagatgctga aggagaccat caacgaggag 1920 gccgccgagt gggaccgcgt gcaccccgtg cacgccggcc ccatcgcccc cggccagatg 1980 cgcgagcccc gcggcagcga catcgccggc accaccagca ccctgcagga gcagatcggc 2040 tggatgacca acaacccccc catccccgtg ggcgagatct acaagcggtg gatcatcctg 2100 ggcctgaaca agatcgtgcg gatgtacagc cccaccagca tcctggacat ccgccagggc 2160 cccaaggagc ccttccgcga ctacgtggac cgcttctaca agaccctgcg cgctgagcag 2220 gccagccagg acgtgaagaa ctggatgacc gagaccctgc tggtgcagaa cgccaacccc 2280 gactgcaaga ccatcctgaa ggctctcggc cccgcggcca ccctggagga gatgatgacc 2340 gcctgccagg gcgtgggcgg ccccggccac aaggcccgcg tgctggccga ggcgatgagc 2400 caggtgacga acccggcgac catcatgatg cagcgcggca acttccgcaa ccagcggaag 2460 accgtcaagt gcttcaactg cggcaaggag ggccacaccg ccaggaactg ccgcgccccc 2520 cgcaagaagg gctgctggcg ctgcggccgc gagggccacc agatgaagga ctgcaccgag 2580 cgccaggcca acttcctggg caagatctgg cccagctaca agggccgccc cggcaacttc 2640 ctgcagagcc gccccgagcc caccgccccc cccgaggaga gcttccgctt cggcgaggag 2700 aagaccaccc ccagccagaa gcaggagccc atcgacaagg agctgtaccc cctgaccagc 2760 ctgcgcagcc tgttcggcaa cgaccccagc agccagtaa 2799 54 5283 DNA Artificial Sequence Description of Artificial Sequence description 54 gtcgacgcca ccatggagcc cgtggacccc cgcctggagc cctggaagca ccccggcagc 60 cagcccaaga ccgccggcac caactgctac tgcaagaagt gctgcttcca ctgccaggtg 120 agcttcatca ccaagggcct gggcatcagc tacggccgca agaagcgccg ccagcgccgc 180 cgcgcccccc ccgacagcga ggtgcaccag gtgagcctgc ccaagcagcc cgccagccag 240 ccccagggcg accccaccgg ccccaaggag agcaagaaga aggtggagcg cgagaccgag 300 accgaccccg tgcaccccgg ggccggccgc agcggcgaca gcgacgagga gctgctgcag 360 accgtgcgct tcatcaagtt cctgtaccag agcaaccccc tgcccagccc caagggcacc 420 cgccaggccg acctgaaccg ccgccgccgc tggcgcgagc gccagcgcca gatccagagc 480 atcagcgcct ggatcatcag cacccacctg ggccgcagca ccgagcccgt gcccctgcag 540 ctgccccccg acctgcgcct gaacctggac tgcagcgagg actgcggcac cagcggcacc 600 cagggcgtgg gcagccccca ggtgctgggc gagagccccg ccgtgctgga cagcggcacc 660 aaggagctcg aggccggcaa gtggagcaag cgcatgagcg gctggagcgc cgtgcgcgag 720 cgcatgaagc gcgccgagcc cgccgagccc gccgccgacg gcgtgggcgc cgtgagccgc 780 gacctggaga agcacggcgc catcaccagc agcaacaccg ccgccaacaa cgccgactgc 840 gcctggctgg aggcccagga ggacgaggac gtgggcttcc ccgtgcgccc ccaggtgccc 900 ctgcgcccca tgacctacaa ggccgccctg gacctgagcc acttcctgaa ggagaagggc 960 ggcctggagg gcctgatcta cagccagaag cgccaggaca tcctggacct gtggatccac 1020 cacacccagg gctacttccc cggctggcag aactacaccc ccggccccgg catccgctac 1080 cccctgacct tcggctggtg cttcaagctg gtgcccgtgg accccgacta cgtggaggag 1140 gccaacgccg gcgagaacaa cagcctgctg caccccatga gccagcacgg catggacgac 1200 cccgagaagg aggtgctggt gtggcgcttc gacagccgcc tggccttcca ccacatggcc 1260 cgcgagctgc accccgagta ctacaaggac tgcctcgagg gcgcccgcgc cagcgtgctg 1320 agcggcggcg agctggacaa gtgggagaag atccgcctgc gccccggcgg caagaagaag 1380 tacaagctga agcacatcgt gtgggccagc cgcgagctgg agcgcttcgc cgtgaacccc 1440 ggcctgctgg agaccagcga gggctgccgc cagatcctgg gccagctgca gcccagcctg 1500 cagaccggca gcgaggagct gcgcagcctg tacaacaccg tggccaccct gtactgcgtg 1560 caccagcgca tcgacgtcaa ggacaccaag gaggccctgg agaagatcga ggaggagcag 1620 aacaagtcca agaagaaggc ccagcaggcc gccgccgccg ccggcaccgg caacagcagc 1680 caggtgagcc agaactaccc catcgtgcag aacctgcagg gccagatggt gcaccaggcc 1740 atcagccccc gcaccctgaa cgcctgggtg aaggtggtgg aggagaaggc cttcagcccc 1800 gaggtgatcc ccatgttcag cgccctgagc gagggcgcca ccccccagga cctgaacacg 1860 atgttgaaca ccgtgggcgg ccaccaggcc gccatgcaga tgctgaagga gaccatcaac 1920 gaggaggccg ccgagtggga ccgcgtgcac cccgtgcacg ccggccccat cgcccccggc 1980 cagatgcgcg agccccgcgg cagcgacatc gccggcacca ccagcaccct gcaggagcag 2040 atcggctgga tgaccaacaa cccccccatc cccgtgggcg agatctacaa gcggtggatc 2100 atcctgggcc tgaacaagat cgtgcggatg tacagcccca ccagcatcct ggacatccgc 2160 cagggcccca aggagccctt ccgcgactac gtggaccgct tctacaagac cctgcgcgct 2220 gagcaggcca gccaggacgt gaagaactgg atgaccgaga ccctgctggt gcagaacgcc 2280 aaccccgact gcaagaccat cctgaaggct ctcggccccg cggccaccct ggaggagatg 2340 atgaccgcct gccagggcgt gggcggcccc ggccacaagg cccgcgtgct ggccgaggcg 2400 atgagccagg tgacgaaccc ggcgaccatc atgatgcagc gcggcaactt ccgcaaccag 2460 cggaagaccg tcaagtgctt caactgcggc aaggagggcc acaccgccag gaactgccgc 2520 gccccccgca agaagggctg ctggcgctgc ggccgcgagg gccaccagat gaaggactgc 2580 accgagcgcc aggccaactt cctgggcaag atctggccca gctacaaggg ccgccccggc 2640 aacttcctgc agagccgccc cgagcccacc gccccccccg aggagagctt ccgcttcggc 2700 gaggagaaga ccacccccag ccagaagcag gagcccatcg acaaggagct gtaccccctg 2760 accagcctgc gcagcctgtt cggcaacgac cccagcagcc agaaagaatt caaggcccgc 2820 gtgctggccg aggcgatgag ccaggtgacg aacccggcga ccatcatgat gcagcgcggc 2880 aacttccgca accagcggaa gaccgtcaag tgcttcaact gcggcaagga gggccacacc 2940 gccaggaact gccgcgcccc ccgcaagaag ggctgctggc gctgcggccg cgaaggacac 3000 caaatgaaag attgcactga gagacaggct aatttcttcc gcgaggacct ggccttcctg 3060 cagggcaagg cccgcgagtt cagcagcgag cagacccgcg ccaacagccc cacccgccgc 3120 gagctgcagg tgtggggcgg cgagaacaac agcctgagcg aggccggcgc cgaccgccag 3180 ggcaccgtga gcttcaactt cccccagatc accctgtggc agcgccccct ggtgaccatc 3240 aggatcggcg gccagctcaa ggaggcgctg ctcgccaccg gcgccgacga caccgtgctg 3300 gaggagatga acctgcccgg caagtggaag cccaagatga tcggcgggat cgggggcttc 3360 atcaaggtgc ggcagtacga ccagatcccc gtggagatct gcggccacaa ggccatcggc 3420 accgtgctgg tgggccccac ccccgtgaac atcatcggcc gcaacctgct gacccagatc 3480 ggctgcaccc tgaacttccc catcagcccc atcgagacgg tgcccgtgaa gctgaagccg 3540 gggatggacg gccccaaggt caagcagtgg cccctgaccg aggagaagat caaggccctg 3600 gtggagatct gcaccgagat ggagaaggag ggcaagatca gcaagatcgg ccccgagaac 3660 ccctacaaca cccccgtgtt cgccatcaag aagaaggaca gcaccaagtg gcgcaagctg 3720 gtggacttcc gcgagctgaa caagcgcacc caggacttct gggaggtgca gctgggcatc 3780 ccccaccccg ccggcctgaa gaagaagaag agcgtgaccg tgctggacgt gggcgacgcc 3840 tacttcagcg tgcccctgga caaggacttc cgcaagtaca ccgccttcac catccccagc 3900 atcaacaacg agacccccgg catccgctac cagtacaacg tgctgcccca gggctggaag 3960 ggcagccccg ccatcttcca gagcagcatg accaagatcc tggagccctt ccgcaagcag 4020 aaccccgaca tcgtgatcta ccaggccccc ctgtacgtgg gcagcgacct ggagatcggc 4080 cagcaccgca ccaagatcga ggagctgcgc cagcacctgc tgcgctgggg cttcaccacc 4140 cccgacaaga agcaccagaa ggagcccccc ttcctgccca tcgagctgca ccccgacaag 4200 tggaccgtgc agcccatcat gctgcccgag aaggacagct ggaccgtgaa cgacatccag 4260 aagctggtgg gcaagctgaa ctgggccagc cagatctacg ccggcatcaa ggtgaagcag 4320 ctgtgcaagc tgctgcgcgg caccaaggcc ctgaccgagg tgatccccct gaccgaggag 4380 gccgagctgg agctggccga gaaccgcgag atcctgaagg agcccgtgca cgaggtgtac 4440 tacgacccca gcaaggacct ggtggccgag atccagaagc agggccaggg ccagtggacc 4500 taccagatct accaggagcc cttcaagaac ctgaagaccg gcaagtacgc ccgcatgcgc 4560 ggcgcccaca ccaacgacgt gaagcagctg accgaggccg tgcagaaggt gagcaccgag 4620 agcatcgtga tctggggcaa gatccccaag ttcaagctgc ccatccagaa ggagacctgg 4680 gaggcctggt ggatggagta ctggcaggcc acctggatcc ccgagtggga gttcgtgaac 4740 accccccccc tggtgaagct gtggtaccag ctggagaagg agcccatcgt gggcgccgag 4800 accttctacg tggacggcgc cgccaaccgc gagaccaagc tgggcaaggc cggctacgtg 4860 accgaccggg gccggcagaa ggtggtgagc atcgccgaca ccaccaacca gaagaccgag 4920 ctgcaggcca tccacctggc cctgcaggac agcggcctgg aggtgaacat cgtgaccgac 4980 agccagtacg ccctgggcat catccaggcc cagcccgaca agagcgagag cgagctggtg 5040 agccagatca tcgagcagct gatcaagaag gagaaggtgt acctggcctg ggtgcccgcc 5100 cacaagggca tcggcggcaa cgagcaggtg gacaagctgg tgagcgccgg catccgcaag 5160 gtgctgttcc tgaacggcat cgatggcggc atcgtgatct accagtacat ggacgacctg 5220 tacgtgggca gcggcggccc taggatcgat taaaagcttc ccggggctag caccggttct 5280 aga 5283 55 4773 DNA Artificial Sequence Description of Artificial Sequence TatRevNefGagProtInaRTmut B 55 gccaccatgg agcccgtgga cccccgcctg gagccctgga agcaccccgg cagccagccc 60 aagaccgccg gcaccaactg ctactgcaag aagtgctgct tccactgcca ggtgagcttc 120 atcaccaagg gcctgggcat cagctacggc cgcaagaagc gccgccagcg ccgccgcgcc 180 ccccccgaca gcgaggtgca ccaggtgagc ctgcccaagc agcccgccag ccagccccag 240 ggcgacccca ccggccccaa ggagagcaag aagaaggtgg agcgcgagac cgagaccgac 300 cccgtgcacc ccggggccgg ccgcagcggc gacagcgacg aggagctgct gcagaccgtg 360 cgcttcatca agttcctgta ccagagcaac cccctgccca gccccaaggg cacccgccag 420 gccgacctga accgccgccg ccgctggcgc gagcgccagc gccagatcca gagcatcagc 480 gcctggatca tcagcaccca cctgggccgc agcaccgagc ccgtgcccct gcagctgccc 540 cccgacctgc gcctgaacct ggactgcagc gaggactgcg gcaccagcgg cacccagggc 600 gtgggcagcc cccaggtgct gggcgagagc cccgccgtgc tggacagcgg caccaaggag 660 ctcgaggccg gcaagtggag caagcgcatg agcggctgga gcgccgtgcg cgagcgcatg 720 aagcgcgccg agcccgccga gcccgccgcc gacggcgtgg gcgccgtgag ccgcgacctg 780 gagaagcacg gcgccatcac cagcagcaac accgccgcca acaacgccga ctgcgcctgg 840 ctggaggccc aggaggacga ggacgtgggc ttccccgtgc gcccccaggt gcccctgcgc 900 cccatgacct acaaggccgc cctggacctg agccacttcc tgaaggagaa gggcggcctg 960 gagggcctga tctacagcca gaagcgccag gacatcctgg acctgtggat ccaccacacc 1020 cagggctact tccccggctg gcagaactac acccccggcc ccggcatccg ctaccccctg 1080 accttcggct ggtgcttcaa gctggtgccc gtggaccccg actacgtgga ggaggccaac 1140 gccggcgaga acaacagcct gctgcacccc atgagccagc acggcatgga cgaccccgag 1200 aaggaggtgc tggtgtggcg cttcgacagc cgcctggcct tccaccacat ggcccgcgag 1260 ctgcaccccg agtactacaa ggactgcaag cttggcgccc gcgccagcgt gctgagcggc 1320 ggcgagctgg acaagtggga gaagatccgc ctgcgccccg gcggcaagaa gaagtacaag 1380 ctgaagcaca tcgtgtgggc cagccgcgag ctggagcgct tcgccgtgaa ccccggcctg 1440 ctggagacca gcgagggctg ccgccagatc ctgggccagc tgcagcccag cctgcagacc 1500 ggcagcgagg agctgcgcag cctgtacaac accgtggcca ccctgtactg cgtgcaccag 1560 cgcatcgacg tcaaggacac caaggaggcc ctggagaaga tcgaggagga gcagaacaag 1620 tccaagaaga aggcccagca ggccgccgcc gccgccggca ccggcaacag cagccaggtg 1680 agccagaact accccatcgt gcagaacctg cagggccaga tggtgcacca ggccatcagc 1740 ccccgcaccc tgaacgcctg ggtgaaggtg gtggaggaga aggccttcag ccccgaggtg 1800 atccccatgt tcagcgccct gagcgagggc gccacccccc aggacctgaa cacgatgttg 1860 aacaccgtgg gcggccacca ggccgccatg cagatgctga aggagaccat caacgaggag 1920 gccgccgagt gggaccgcgt gcaccccgtg cacgccggcc ccatcgcccc cggccagatg 1980 cgcgagcccc gcggcagcga catcgccggc accaccagca ccctgcagga gcagatcggc 2040 tggatgacca acaacccccc catccccgtg ggcgagatct acaagcggtg gatcatcctg 2100 ggcctgaaca agatcgtgcg gatgtacagc cccaccagca tcctggacat ccgccagggc 2160 cccaaggagc ccttccgcga ctacgtggac cgcttctaca agaccctgcg cgctgagcag 2220 gccagccagg acgtgaagaa ctggatgacc gagaccctgc tggtgcagaa cgccaacccc 2280 gactgcaaga ccatcctgaa ggctctcggc cccgcggcca ccctggagga gatgatgacc 2340 gcctgccagg gcgtgggcgg ccccggccac aaggcccgcg tgctggccga ggcgatgagc 2400 caggtgacga acccggcgac catcatgatg cagcgcggca acttccgcaa ccagcggaag 2460 accgtcaagt gcttcaactg cggcaaggag ggccacaccg ccaggaactg ccgcgccccc 2520 cgcaagaagg gctgctggcg ctgcggccgc gagggccacc agatgaagga ctgcaccgag 2580 cgccaggcca acttcctggg caagatctgg cccagctaca agggccgccc cggcaacttc 2640 ctgcagagcc gccccgagcc caccgccccc cccgaggaga gcttccgctt cggcgaggag 2700 aagaccaccc ccagccagaa gcaggagccc atcgacaagg agctgtaccc cctgaccagc 2760 ctgcgcagcc tgttcggcaa cgaccccagc agccagaaag aattccccca gatcaccctg 2820 tggcagcgcc ccctggtgac catcaggatc ggcggccagc tcaaggaggc gctgctcgcc 2880 accggcgccg acgacaccgt gctggaggag atgaacctgc ccggcaagtg gaagcccaag 2940 atgatcggcg ggatcggggg cttcatcaag gtgcggcagt acgaccagat ccccgtggag 3000 atctgcggcc acaaggccat cggcaccgtg ctggtgggcc ccacccccgt gaacatcatc 3060 ggccgcaacc tgctgaccca gatcggctgc accctgaact tccccatcag ccccatcgag 3120 acggtgcccg tgaagctgaa gccggggatg gacggcccca aggtcaagca gtggcccctg 3180 accgaggaga agatcaaggc cctggtggag atctgcaccg agatggagaa ggagggcaag 3240 atcagcaaga tcggccccga gaacccctac aacacccccg tgttcgccat caagaagaag 3300 gacagcacca agtggcgcaa gctggtggac ttccgcgagc tgaacaagcg cacccaggac 3360 ttctgggagg tgcagctggg catcccccac cccgccggcc tgaagaagaa gaagagcgtg 3420 accgtgctgg acgtgggcga cgcctacttc agcgtgcccc tggacaagga cttccgcaag 3480 tacaccgcct tcaccatccc cagcatcaac aacgagaccc ccggcatccg ctaccagtac 3540 aacgtgctgc cccagggctg gaagggcagc cccgccatct tccagagcag catgaccaag 3600 atcctggagc ccttccgcaa gcagaacccc gacatcgtga tctaccaggc ccccctgtac 3660 gtgggcagcg acctggagat cggccagcac cgcaccaaga tcgaggagct gcgccagcac 3720 ctgctgcgct ggggcttcac cacccccgac aagaagcacc agaaggagcc ccccttcctg 3780 cccatcgagc tgcaccccga caagtggacc gtgcagccca tcatgctgcc cgagaaggac 3840 agctggaccg tgaacgacat ccagaagctg gtgggcaagc tgaactgggc cagccagatc 3900 tacgccggca tcaaggtgaa gcagctgtgc aagctgctgc gcggcaccaa ggccctgacc 3960 gaggtgatcc ccctgaccga ggaggccgag ctggagctgg ccgagaaccg cgagatcctg 4020 aaggagcccg tgcacgaggt gtactacgac cccagcaagg acctggtggc cgagatccag 4080 aagcagggcc agggccagtg gacctaccag atctaccagg agcccttcaa gaacctgaag 4140 accggcaagt acgcccgcat gcgcggcgcc cacaccaacg acgtgaagca gctgaccgag 4200 gccgtgcaga aggtgagcac cgagagcatc gtgatctggg gcaagatccc caagttcaag 4260 ctgcccatcc agaaggagac ctgggaggcc tggtggatgg agtactggca ggccacctgg 4320 atccccgagt gggagttcgt gaacaccccc cccctggtga agctgtggta ccagctggag 4380 aaggagccca tcgtgggcgc cgagaccttc tacgtggacg gcgccgccaa ccgcgagacc 4440 aagctgggca aggccggcta cgtgaccgac cggggccggc agaaggtggt gagcatcgcc 4500 gacaccacca accagaagac cgagctgcag gccatccacc tggccctgca ggacagcggc 4560 ctggaggtga acatcgtgac cgacagccag tacgccctgg gcatcatcca ggcccagccc 4620 gacaagagcg agagcgagct ggtgagccag atcatcgagc agctgatcaa gaaggagaag 4680 gtgtacctgg cctgggtgcc cgcccacaag ggcatcggcg gcaacgagca ggtggacaag 4740 ctggtgagcg ccggcatccg caaggtgctc taa 4773 56 3636 DNA Artificial Sequence Description of Artificial Sequence TatRevNefp2Pol.opt_B 56 gccaccatgg agcccgtgga cccccgcctg gagccctgga agcaccccgg cagccagccc 60 aagaccgccg gcaccaactg ctactgcaag aagtgctgct tccactgcca ggtgagcttc 120 atcaccaagg gcctgggcat cagctacggc cgcaagaagc gccgccagcg ccgccgcgcc 180 ccccccgaca gcgaggtgca ccaggtgagc ctgcccaagc agcccgccag ccagccccag 240 ggcgacccca ccggccccaa ggagagcaag aagaaggtgg agcgcgagac cgagaccgac 300 cccgtgcacc ccggggccgg ccgcagcggc gacagcgacg aggagctgct gcagaccgtg 360 cgcttcatca agttcctgta ccagagcaac cccctgccca gccccaaggg cacccgccag 420 gccgacctga accgccgccg ccgctggcgc gagcgccagc gccagatcca gagcatcagc 480 gcctggatca tcagcaccca cctgggccgc agcaccgagc ccgtgcccct gcagctgccc 540 cccgacctgc gcctgaacct ggactgcagc gaggactgcg gcaccagcgg cacccagggc 600 gtgggcagcc cccaggtgct gggcgagagc cccgccgtgc tggacagcgg caccaaggag 660 ctcgaggccg gcaagtggag caagcgcatg agcggctgga gcgccgtgcg cgagcgcatg 720 aagcgcgccg agcccgccga gcccgccgcc gacggcgtgg gcgccgtgag ccgcgacctg 780 gagaagcacg gcgccatcac cagcagcaac accgccgcca acaacgccga ctgcgcctgg 840 ctggaggccc aggaggacga ggacgtgggc ttccccgtgc gcccccaggt gcccctgcgc 900 cccatgacct acaaggccgc cctggacctg agccacttcc tgaaggagaa gggcggcctg 960 gagggcctga tctacagcca gaagcgccag gacatcctgg acctgtggat ccaccacacc 1020 cagggctact tccccggctg gcagaactac acccccggcc ccggcatccg ctaccccctg 1080 accttcggct ggtgcttcaa gctggtgccc gtggaccccg actacgtgga ggaggccaac 1140 gccggcgaga acaacagcct gctgcacccc atgagccagc acggcatgga cgaccccgag 1200 aaggaggtgc tggtgtggcg cttcgacagc cgcctggcct tccaccacat ggcccgcgag 1260 ctgcaccccg agtactacaa ggactgcgaa ttcgccgagg cgatgagcca ggtgacgaac 1320 ccggcgacca tcatgatgca gcgcggcaac ttccgcaacc agcggaagac cgtcaagtgc 1380 ttcaactgcg gcaaggaggg ccacaccgcc aggaactgcc gcgccccccg caagaagggc 1440 tgctggcgct gcggccgcga aggacaccaa atgaaagatt gcactgagag acaggctaat 1500 ttcttccgcg aggacctggc cttcctgcag ggcaaggccc gcgagttcag cagcgagcag 1560 acccgcgcca acagccccac ccgccgcgag ctgcaggtgt ggggcggcga gaacaacagc 1620 ctgagcgagg ccggcgccga ccgccagggc accgtgagct tcaacttccc ccagatcacc 1680 ctgtggcagc gccccctggt gaccatcagg atcggcggcc

agctcaagga ggcgctgctc 1740 gccaccggcg ccgacgacac cgtgctggag gagatgaacc tgcccggcaa gtggaagccc 1800 aagatgatcg gcgggatcgg gggcttcatc aaggtgcggc agtacgacca gatccccgtg 1860 gagatctgcg gccacaaggc catcggcacc gtgctggtgg gccccacccc cgtgaacatc 1920 atcggccgca acctgctgac ccagatcggc tgcaccctga acttccccat cagccccatc 1980 gagacggtgc ccgtgaagct gaagccgggg atggacggcc ccaaggtcaa gcagtggccc 2040 ctgaccgagg agaagatcaa ggccctggtg gagatctgca ccgagatgga gaaggagggc 2100 aagatcagca agatcggccc cgagaacccc tacaacaccc ccgtgttcgc catcaagaag 2160 aaggacagca ccaagtggcg caagctggtg gacttccgcg agctgaacaa gcgcacccag 2220 gacttctggg aggtgcagct gggcatcccc caccccgccg gcctgaagaa gaagaagagc 2280 gtgaccgtgc tggacgtggg cgacgcctac ttcagcgtgc ccctggacaa ggacttccgc 2340 aagtacaccg ccttcaccat ccccagcatc aacaacgaga cccccggcat ccgctaccag 2400 tacaacgtgc tgccccaggg ctggaagggc agccccgcca tcttccagag cagcatgacc 2460 aagatcctgg agcccttccg caagcagaac cccgacatcg tgatctacca ggcccccctg 2520 tacgtgggca gcgacctgga gatcggccag caccgcacca agatcgagga gctgcgccag 2580 cacctgctgc gctggggctt caccaccccc gacaagaagc accagaagga gccccccttc 2640 ctgcccatcg agctgcaccc cgacaagtgg accgtgcagc ccatcatgct gcccgagaag 2700 gacagctgga ccgtgaacga catccagaag ctggtgggca agctgaactg ggccagccag 2760 atctacgccg gcatcaaggt gaagcagctg tgcaagctgc tgcgcggcac caaggccctg 2820 accgaggtga tccccctgac cgaggaggcc gagctggagc tggccgagaa ccgcgagatc 2880 ctgaaggagc ccgtgcacga ggtgtactac gaccccagca aggacctggt ggccgagatc 2940 cagaagcagg gccagggcca gtggacctac cagatctacc aggagccctt caagaacctg 3000 aagaccggca agtacgcccg catgcgcggc gcccacacca acgacgtgaa gcagctgacc 3060 gaggccgtgc agaaggtgag caccgagagc atcgtgatct ggggcaagat ccccaagttc 3120 aagctgccca tccagaagga gacctgggag gcctggtgga tggagtactg gcaggccacc 3180 tggatccccg agtgggagtt cgtgaacacc ccccccctgg tgaagctgtg gtaccagctg 3240 gagaaggagc ccatcgtggg cgccgagacc ttctacgtgg acggcgccgc caaccgcgag 3300 accaagctgg gcaaggccgg ctacgtgacc gaccggggcc ggcagaaggt ggtgagcatc 3360 gccgacacca ccaaccagaa gaccgagctg caggccatcc acctggccct gcaggacagc 3420 ggcctggagg tgaacatcgt gaccgacagc cagtacgccc tgggcatcat ccaggcccag 3480 cccgacaaga gcgagagcga gctggtgagc cagatcatcg agcagctgat caagaaggag 3540 aaggtgtacc tggcctgggt gcccgcccac aagggcatcg gcggcaacga gcaggtggac 3600 aagctggtga gcgccggcat ccgcaaggtg ctgtaa 3636 57 3261 DNA Artificial Sequence Description of Artificial Sequence TatRevNefprotRTopt B 57 gccaccatgg agcccgtgga cccccgcctg gagccctgga agcaccccgg cagccagccc 60 aagaccgccg gcaccaactg ctactgcaag aagtgctgct tccactgcca ggtgagcttc 120 atcaccaagg gcctgggcat cagctacggc cgcaagaagc gccgccagcg ccgccgcgcc 180 ccccccgaca gcgaggtgca ccaggtgagc ctgcccaagc agcccgccag ccagccccag 240 ggcgacccca ccggccccaa ggagagcaag aagaaggtgg agcgcgagac cgagaccgac 300 cccgtgcacc ccggggccgg ccgcagcggc gacagcgacg aggagctgct gcagaccgtg 360 cgcttcatca agttcctgta ccagagcaac cccctgccca gccccaaggg cacccgccag 420 gccgacctga accgccgccg ccgctggcgc gagcgccagc gccagatcca gagcatcagc 480 gcctggatca tcagcaccca cctgggccgc agcaccgagc ccgtgcccct gcagctgccc 540 cccgacctgc gcctgaacct ggactgcagc gaggactgcg gcaccagcgg cacccagggc 600 gtgggcagcc cccaggtgct gggcgagagc cccgccgtgc tggacagcgg caccaaggag 660 ctcgaggccg gcaagtggag caagcgcatg agcggctgga gcgccgtgcg cgagcgcatg 720 aagcgcgccg agcccgccga gcccgccgcc gacggcgtgg gcgccgtgag ccgcgacctg 780 gagaagcacg gcgccatcac cagcagcaac accgccgcca acaacgccga ctgcgcctgg 840 ctggaggccc aggaggacga ggacgtgggc ttccccgtgc gcccccaggt gcccctgcgc 900 cccatgacct acaaggccgc cctggacctg agccacttcc tgaaggagaa gggcggcctg 960 gagggcctga tctacagcca gaagcgccag gacatcctgg acctgtggat ccaccacacc 1020 cagggctact tccccggctg gcagaactac acccccggcc ccggcatccg ctaccccctg 1080 accttcggct ggtgcttcaa gctggtgccc gtggaccccg actacgtgga ggaggccaac 1140 gccggcgaga acaacagcct gctgcacccc atgagccagc acggcatgga cgaccccgag 1200 aaggaggtgc tggtgtggcg cttcgacagc cgcctggcct tccaccacat ggcccgcgag 1260 ctgcaccccg agtactacaa ggactgcgaa ttcccccaga tcaccctgtg gcagcgcccc 1320 ctggtgacca tcaggatcgg cggccagctc aaggaggcgc tgctcgccac cggcgccgac 1380 gacaccgtgc tggaggagat gaacctgccc ggcaagtgga agcccaagat gatcggcggg 1440 atcgggggct tcatcaaggt gcggcagtac gaccagatcc ccgtggagat ctgcggccac 1500 aaggccatcg gcaccgtgct ggtgggcccc acccccgtga acatcatcgg ccgcaacctg 1560 ctgacccaga tcggctgcac cctgaacttc cccatcagcc ccatcgagac ggtgcccgtg 1620 aagctgaagc cggggatgga cggccccaag gtcaagcagt ggcccctgac cgaggagaag 1680 atcaaggccc tggtggagat ctgcaccgag atggagaagg agggcaagat cagcaagatc 1740 ggccccgaga acccctacaa cacccccgtg ttcgccatca agaagaagga cagcaccaag 1800 tggcgcaagc tggtggactt ccgcgagctg aacaagcgca cccaggactt ctgggaggtg 1860 cagctgggca tcccccaccc cgccggcctg aagaagaaga agagcgtgac cgtgctggac 1920 gtgggcgacg cctacttcag cgtgcccctg gacaaggact tccgcaagta caccgccttc 1980 accatcccca gcatcaacaa cgagaccccc ggcatccgct accagtacaa cgtgctgccc 2040 cagggctgga agggcagccc cgccatcttc cagagcagca tgaccaagat cctggagccc 2100 ttccgcaagc agaaccccga catcgtgatc taccaggccc ccctgtacgt gggcagcgac 2160 ctggagatcg gccagcaccg caccaagatc gaggagctgc gccagcacct gctgcgctgg 2220 ggcttcacca cccccgacaa gaagcaccag aaggagcccc ccttcctgcc catcgagctg 2280 caccccgaca agtggaccgt gcagcccatc atgctgcccg agaaggacag ctggaccgtg 2340 aacgacatcc agaagctggt gggcaagctg aactgggcca gccagatcta cgccggcatc 2400 aaggtgaagc agctgtgcaa gctgctgcgc ggcaccaagg ccctgaccga ggtgatcccc 2460 ctgaccgagg aggccgagct ggagctggcc gagaaccgcg agatcctgaa ggagcccgtg 2520 cacgaggtgt actacgaccc cagcaaggac ctggtggccg agatccagaa gcagggccag 2580 ggccagtgga cctaccagat ctaccaggag cccttcaaga acctgaagac cggcaagtac 2640 gcccgcatgc gcggcgccca caccaacgac gtgaagcagc tgaccgaggc cgtgcagaag 2700 gtgagcaccg agagcatcgt gatctggggc aagatcccca agttcaagct gcccatccag 2760 aaggagacct gggaggcctg gtggatggag tactggcagg ccacctggat ccccgagtgg 2820 gagttcgtga acaccccccc cctggtgaag ctgtggtacc agctggagaa ggagcccatc 2880 gtgggcgccg agaccttcta cgtggacggc gccgccaacc gcgagaccaa gctgggcaag 2940 gccggctacg tgaccgaccg gggccggcag aaggtggtga gcatcgccga caccaccaac 3000 cagaagaccg agctgcaggc catccacctg gccctgcagg acagcggcct ggaggtgaac 3060 atcgtgaccg acagccagta cgccctgggc atcatccagg cccagcccga caagagcgag 3120 agcgagctgg tgagccagat catcgagcag ctgatcaaga aggagaaggt gtacctggcc 3180 tgggtgcccg cccacaaggg catcggcggc aacgagcagg tggacaagct ggtgagcgcc 3240 ggcatccgca aggtgctcta a 3261 58 576 DNA Artificial Sequence Description of Artificial Sequence vif.opt.SF2 58 atggagaacc gctggcaggt gatgatcgtg tggcaggtgg accgcatgcg catccgcacc 60 tggaagagcc tggtgaagca ccacatgtac atcagcaaga aggccaaggg ctggttctac 120 cgccaccact acgagagcac ccacccccgc gtgagcagcg aggtgcacat ccccctgggc 180 gacgccaagc tggtgatcac cacctactgg ggcctgcaca ccggcgagcg cgagtggcac 240 ctgggccagg gcgtggccat cgagtggcgc aagaagaagt acagcaccca ggtggacccc 300 ggcctggccg accagctgat ccacctgcac tacttcgact gcttcagcga gagcgccatc 360 aagaacgcca tcctgggcta ccgcgtgagc ccccgctgcg agtaccaggc cggccacaac 420 aaggtgggca gcctgcagta cctggccctg gccgccctga tcacccccaa gaagaccaag 480 ccccccctgc ccagcgtgaa gaagctgacc gaggaccgct ggaacaagcc ccagaagacc 540 aagggccacc gcggcagcca caccatgaac ggccac 576 59 291 DNA Artificial Sequence Description of Artificial Sequence vpr.opt.SF2 59 atggagcagg cccccgagga ccagggcccc cagcgcgagc cctacaacga gtggaccctg 60 gagctgctgg aggagctgaa gcgcgaggcc gtgcgccact tcccccgccc ctggctgcac 120 agcctgggcc agtacatcta cgagacctac ggcgacacct gggccggcgt ggaggccatc 180 atccgcatcc tgcagcagct gctgttcatc cacttccgca tcggctgcca gcacagccgc 240 atcggcatca tccagcagcg ccgcgcccgc cgcaacggcg ccagccgcag c 291 60 243 DNA Artificial Sequence Description of Artificial Sequence vpu.opt.SF162 60 atgcagcccc tgcagatcct ggccatcgtg gccctggtgg tggccgccat catcgccatc 60 gtggtgtgga ccatcgtgta catcgagtac cgcaagatcc tgcgccagcg caagatcgac 120 cgcctgatcg accgcatcac cgagcgcgcc gaggacagcg gcaacgagag cgagggcgac 180 caggaggagc tgagcgccct ggtggagcgc ggccacctgg ccccctggga cgtggacgac 240 ctg 243 61 2007 DNA Artificial Sequence Description of Artificial Sequence gp140modSF162.GM135-154-186-195 61 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccagcg ccgtggagaa gctgtgggtg accgtgtact acggcgtgcc cgtgtggaag 120 gaggccacca ccaccctgtt ctgcgccagc gacgccaagg cctacgacac cgaggtgcac 180 aacgtgtggg ccacccacgc ctgcgtgccc accgacccca acccccagga gatcgtgctg 240 gagaacgtga ccgagaactt caacatgtgg aagaacaaca tggtggagca gatgcacgag 300 gacatcatca gcctgtggga ccagagcctg aagccctgcg tgaagctgac ccccctgtgc 360 gtgaccctgc actgcaccaa cctgaagcag gccaccaaca ccaagagcag caactggaag 420 gagatggacc gcggcgagat caagcagtgc agcttcaagg tgaccaccag catccgcaac 480 aagatgcaga aggagtacgc cctgttctac aagctggacg tggtgcccat cgacaacgac 540 cagaccagct acaagctgat caactgccag accagcgtga tcacccaggc ctgccccaag 600 gtgagcttcg agcccatccc catccactac tgcgcccccg ccggcttcgc catcctgaag 660 tgcaacgaca agaagttcaa cggcagcggc ccctgcacca acgtgagcac cgtgcagtgc 720 acccacggca tccgccccgt ggtgagcacc cagctgctgc tgaacggcag cctggccgag 780 gagggcgtgg tgatccgcag cgagaacttc accgacaacg ccaagaccat catcgtgcag 840 ctgaaggaga gcgtggagat caactgcacc cgccccaaca acaacacccg caagagcatc 900 accatcggcc ccggccgcgc cttctacgcc accggcgaca tcatcggcga catccgccag 960 gcccactgca acatcagcgg cgagaagtgg aacaacaccc tgaagcagat cgtgaccaag 1020 ctgcaggccc agttcggcaa caagaccatc gtgttcaagc agagcagcgg cggcgacccc 1080 gagatcgtga tgcacagctt caactgcggc ggcgagttct tctactgcaa cagcacccag 1140 ctgttcaaca gcacctggaa caacaccatc ggccccaaca acaccaacgg caccatcacc 1200 ctgccctgcc gcatcaagca gatcatcaac cgctggcagg aggtgggcaa ggccatgtac 1260 gcccccccca tccgcggcca gatccgctgc agcagcaaca tcaccggcct gctgctgacc 1320 cgcgacggcg gcaaggagat cagcaacacc accgagatct tccgccccgg cggcggcgac 1380 atgcgcgaca actggcgcag cgagctgtac aagtacaagg tggtgaagat cgagcccctg 1440 ggcgtggccc ccaccaaggc caagcgccgc gtggtgcagc gcgagaagcg cgccgtgacc 1500 ctgggcgcca tgttcctggg cttcctgggc gccgccggca gcaccatggg cgcccgcagc 1560 ctgaccctga ccgtgcaggc ccgccagctg ctgagcggca tcgtgcagca gcagaacaac 1620 ctgctgcgcg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag 1680 cagctgcagg cccgcgtgct ggccgtggag cgctacctga aggaccagca gctgctgggc 1740 atctggggct gcagcggcaa gctgatctgc accaccgccg tgccctggaa cgccagctgg 1800 agcaacaaga gcctggacca gatctggaac aacatgacct ggatggagtg ggagcgcgag 1860 atcgacaact acaccaacct gatctacacc ctgatcgagg agagccagaa ccagcaggag 1920 aagaacgagc aggagctgct ggagctggac aagtgggcca gcctgtggaa ctggttcgac 1980 atcagcaagt ggctgtggta catctaa 2007 62 2007 DNA Artificial Sequence Description of Artificial Sequence gp140modSF162.GM154 62 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccagcg ccgtggagaa gctgtgggtg accgtgtact acggcgtgcc cgtgtggaag 120 gaggccacca ccaccctgtt ctgcgccagc gacgccaagg cctacgacac cgaggtgcac 180 aacgtgtggg ccacccacgc ctgcgtgccc accgacccca acccccagga gatcgtgctg 240 gagaacgtga ccgagaactt caacatgtgg aagaacaaca tggtggagca gatgcacgag 300 gacatcatca gcctgtggga ccagagcctg aagccctgcg tgaagctgac ccccctgtgc 360 gtgaccctgc actgcaccaa cctgaagaac gccaccaaca ccaagagcag caactggaag 420 gagatggacc gcggcgagat caagcagtgc agcttcaagg tgaccaccag catccgcaac 480 aagatgcaga aggagtacgc cctgttctac aagctggacg tggtgcccat cgacaacgac 540 aacaccagct acaagctgat caactgcaac accagcgtga tcacccaggc ctgccccaag 600 gtgagcttcg agcccatccc catccactac tgcgcccccg ccggcttcgc catcctgaag 660 tgcaacgaca agaagttcaa cggcagcggc ccctgcacca acgtgagcac cgtgcagtgc 720 acccacggca tccgccccgt ggtgagcacc cagctgctgc tgaacggcag cctggccgag 780 gagggcgtgg tgatccgcag cgagaacttc accgacaacg ccaagaccat catcgtgcag 840 ctgaaggaga gcgtggagat caactgcacc cgccccaaca acaacacccg caagagcatc 900 accatcggcc ccggccgcgc cttctacgcc accggcgaca tcatcggcga catccgccag 960 gcccactgca acatcagcgg cgagaagtgg aacaacaccc tgaagcagat cgtgaccaag 1020 ctgcaggccc agttcggcaa caagaccatc gtgttcaagc agagcagcgg cggcgacccc 1080 gagatcgtga tgcacagctt caactgcggc ggcgagttct tctactgcaa cagcacccag 1140 ctgttcaaca gcacctggaa caacaccatc ggccccaaca acaccaacgg caccatcacc 1200 ctgccctgcc gcatcaagca gatcatcaac cgctggcagg aggtgggcaa ggccatgtac 1260 gcccccccca tccgcggcca gatccgctgc agcagcaaca tcaccggcct gctgctgacc 1320 cgcgacggcg gcaaggagat cagcaacacc accgagatct tccgccccgg cggcggcgac 1380 atgcgcgaca actggcgcag cgagctgtac aagtacaagg tggtgaagat cgagcccctg 1440 ggcgtggccc ccaccaaggc caagcgccgc gtggtgcagc gcgagaagcg cgccgtgacc 1500 ctgggcgcca tgttcctggg cttcctgggc gccgccggca gcaccatggg cgcccgcagc 1560 ctgaccctga ccgtgcaggc ccgccagctg ctgagcggca tcgtgcagca gcagaacaac 1620 ctgctgcgcg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag 1680 cagctgcagg cccgcgtgct ggccgtggag cgctacctga aggaccagca gctgctgggc 1740 atctggggct gcagcggcaa gctgatctgc accaccgccg tgccctggaa cgccagctgg 1800 agcaacaaga gcctggacca gatctggaac aacatgacct ggatggagtg ggagcgcgag 1860 atcgacaact acaccaacct gatctacacc ctgatcgagg agagccagaa ccagcaggag 1920 aagaacgagc aggagctgct ggagctggac aagtgggcca gcctgtggaa ctggttcgac 1980 atcagcaagt ggctgtggta catctaa 2007 63 2007 DNA Artificial Sequence Description of Artificial Sequence gp140modSF162.GM154-186-195 63 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccagcg ccgtggagaa gctgtgggtg accgtgtact acggcgtgcc cgtgtggaag 120 gaggccacca ccaccctgtt ctgcgccagc gacgccaagg cctacgacac cgaggtgcac 180 aacgtgtggg ccacccacgc ctgcgtgccc accgacccca acccccagga gatcgtgctg 240 gagaacgtga ccgagaactt caacatgtgg aagaacaaca tggtggagca gatgcacgag 300 gacatcatca gcctgtggga ccagagcctg aagccctgcg tgaagctgac ccccctgtgc 360 gtgaccctgc actgcaccaa cctgaagaac gccaccaaca ccaagagcag caactggaag 420 gagatggacc gcggcgagat caagcagtgc agcttcaagg tgaccaccag catccgcaac 480 aagatgcaga aggagtacgc cctgttctac aagctggacg tggtgcccat cgacaacgac 540 cagaccagct acaagctgat caactgccag accagcgtga tcacccaggc ctgccccaag 600 gtgagcttcg agcccatccc catccactac tgcgcccccg ccggcttcgc catcctgaag 660 tgcaacgaca agaagttcaa cggcagcggc ccctgcacca acgtgagcac cgtgcagtgc 720 acccacggca tccgccccgt ggtgagcacc cagctgctgc tgaacggcag cctggccgag 780 gagggcgtgg tgatccgcag cgagaacttc accgacaacg ccaagaccat catcgtgcag 840 ctgaaggaga gcgtggagat caactgcacc cgccccaaca acaacacccg caagagcatc 900 accatcggcc ccggccgcgc cttctacgcc accggcgaca tcatcggcga catccgccag 960 gcccactgca acatcagcgg cgagaagtgg aacaacaccc tgaagcagat cgtgaccaag 1020 ctgcaggccc agttcggcaa caagaccatc gtgttcaagc agagcagcgg cggcgacccc 1080 gagatcgtga tgcacagctt caactgcggc ggcgagttct tctactgcaa cagcacccag 1140 ctgttcaaca gcacctggaa caacaccatc ggccccaaca acaccaacgg caccatcacc 1200 ctgccctgcc gcatcaagca gatcatcaac cgctggcagg aggtgggcaa ggccatgtac 1260 gcccccccca tccgcggcca gatccgctgc agcagcaaca tcaccggcct gctgctgacc 1320 cgcgacggcg gcaaggagat cagcaacacc accgagatct tccgccccgg cggcggcgac 1380 atgcgcgaca actggcgcag cgagctgtac aagtacaagg tggtgaagat cgagcccctg 1440 ggcgtggccc ccaccaaggc caagcgccgc gtggtgcagc gcgagaagcg cgccgtgacc 1500 ctgggcgcca tgttcctggg cttcctgggc gccgccggca gcaccatggg cgcccgcagc 1560 ctgaccctga ccgtgcaggc ccgccagctg ctgagcggca tcgtgcagca gcagaacaac 1620 ctgctgcgcg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag 1680 cagctgcagg cccgcgtgct ggccgtggag cgctacctga aggaccagca gctgctgggc 1740 atctggggct gcagcggcaa gctgatctgc accaccgccg tgccctggaa cgccagctgg 1800 agcaacaaga gcctggacca gatctggaac aacatgacct ggatggagtg ggagcgcgag 1860 atcgacaact acaccaacct gatctacacc ctgatcgagg agagccagaa ccagcaggag 1920 aagaacgagc aggagctgct ggagctggac aagtgggcca gcctgtggaa ctggttcgac 1980 atcagcaagt ggctgtggta catctaa 2007 64 2007 DNA Artificial Sequence Description of Artificial Sequence gp140mut7.modSF162.GM154 64 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60 tcgcccagcg ccgtggagaa gctgtgggtg accgtgtact acggcgtgcc cgtgtggaag 120 gaggccacca ccaccctgtt ctgcgccagc gacgccaagg cctacgacac cgaggtgcac 180 aacgtgtggg ccacccacgc ctgcgtgccc accgacccca acccccagga gatcgtgctg 240 gagaacgtga ccgagaactt caacatgtgg aagaacaaca tggtggagca gatgcacgag 300 gacatcatca gcctgtggga ccagagcctg aagccctgcg tgaagctgac ccccctgtgc 360 gtgaccctgc actgcaccaa cctgaagaac gccaccaaca ccaagagcag caactggaag 420 gagatggacc gcggcgagat caagcagtgc agcttcaagg tgaccaccag catccgcaac 480 aagatgcaga aggagtacgc cctgttctac aagctggacg tggtgcccat cgacaacgac 540 aacaccagct acaagctgat caactgcaac accagcgtga tcacccaggc ctgccccaag 600 gtgagcttcg agcccatccc catccactac tgcgcccccg ccggcttcgc catcctgaag 660 tgcaacgaca agaagttcaa cggcagcggc ccctgcacca acgtgagcac cgtgcagtgc 720 acccacggca tccgccccgt ggtgagcacc cagctgctgc tgaacggcag cctggccgag 780 gagggcgtgg tgatccgcag cgagaacttc accgacaacg ccaagaccat catcgtgcag 840 ctgaaggaga gcgtggagat caactgcacc cgccccaaca acaacacccg caagagcatc 900 accatcggcc ccggccgcgc cttctacgcc accggcgaca tcatcggcga catccgccag 960 gcccactgca acatcagcgg cgagaagtgg aacaacaccc tgaagcagat cgtgaccaag 1020 ctgcaggccc agttcggcaa caagaccatc gtgttcaagc agagcagcgg cggcgacccc 1080 gagatcgtga tgcacagctt caactgcggc ggcgagttct tctactgcaa cagcacccag 1140 ctgttcaaca gcacctggaa caacaccatc ggccccaaca acaccaacgg caccatcacc 1200 ctgccctgcc gcatcaagca gatcatcaac cgctggcagg aggtgggcaa ggccatgtac 1260 gcccccccca tccgcggcca gatccgctgc agcagcaaca tcaccggcct gctgctgacc 1320 cgcgacggcg gcaaggagat cagcaacacc accgagatct tccgccccgg cggcggcgac 1380 atgcgcgaca actggcgcag cgagctgtac aagtacaagg tggtgaagat cgagcccctg 1440 ggcgtggccc ccaccaaggc catcagcagc gtggtgcaga gcgagaagag cgccgtgacc 1500 ctgggcgcca tgttcctggg cttcctgggc gccgccggca gcaccatggg cgcccgcagc 1560 ctgaccctga ccgtgcaggc ccgccagctg ctgagcggca tcgtgcagca gcagaacaac 1620 ctgctgcgcg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag 1680 cagctgcagg cccgcgtgct ggccgtggag cgctacctga aggaccagca gctgctgggc 1740 atctggggct gcagcggcaa gctgatctgc accaccgccg tgccctggaa cgccagctgg 1800

agcaacaaga gcctggacca gatctggaac aacatgacct ggatggagtg ggagcgcgag 1860 atcgacaact acaccaacct gatctacacc ctgatcgagg agagccagaa ccagcaggag 1920 aagaacgagc aggagctgct ggagctggac aagtgggcca gcctgtggaa ctggttcgac 1980 atcagcaagt ggctgtggta catctaa 2007 65 100 PRT Artificial Sequence Description of Artificial Sequence gp140modSF162 65 Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu 1 5 10 15 Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Leu Lys Asn Ala Thr 20 25 30 Asn Thr Lys Ser Ser Asn Trp Lys Glu Met Asp Arg Gly Glu Ile Lys 35 40 45 Asn Cys Ser Phe Lys Val Thr Thr Ser Ile Arg Asn Lys Met Gln Lys 50 55 60 Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Asp 65 70 75 80 Asn Thr Ser Tyr Lys Leu Ile Asn Cys Asn Thr Ser Val Ile Thr Gln 85 90 95 Ala Cys Pro Lys 100 66 100 PRT Artificial Sequence Description of Artificial Sequence gp140.modSF162.GM154 66 Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu 1 5 10 15 Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Leu Lys Asn Ala Thr 20 25 30 Asn Thr Lys Ser Ser Asn Trp Lys Glu Met Asp Arg Gly Glu Ile Lys 35 40 45 Gln Cys Ser Phe Lys Val Thr Thr Ser Ile Arg Asn Lys Met Gln Lys 50 55 60 Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Asp 65 70 75 80 Asn Thr Ser Tyr Lys Leu Ile Asn Cys Asn Thr Ser Val Ile Thr Gln 85 90 95 Ala Cys Pro Lys 100 67 100 PRT Artificial Sequence Description of Artificial Sequence gp140.modSF162.GM154-186-195 67 Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu 1 5 10 15 Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Leu Lys Asn Ala Thr 20 25 30 Asn Thr Lys Ser Ser Asn Trp Lys Glu Met Asp Arg Gly Glu Ile Lys 35 40 45 Gln Cys Ser Phe Lys Val Thr Thr Ser Ile Arg Asn Lys Met Gln Lys 50 55 60 Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Asp 65 70 75 80 Gln Thr Ser Tyr Lys Leu Ile Asn Cys Gln Thr Ser Val Ile Thr Gln 85 90 95 Ala Cys Pro Lys 100 68 100 PRT Artificial Sequence Description of Artificial Sequence gp140.modSF162.GM135-154-186-195 68 Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu 1 5 10 15 Thr Pro Leu Cys Val Thr Leu His Cys Thr Asn Leu Lys Gln Ala Thr 20 25 30 Asn Thr Lys Ser Ser Asn Trp Lys Glu Met Asp Arg Gly Glu Ile Lys 35 40 45 Gln Cys Ser Phe Lys Val Thr Thr Ser Ile Arg Asn Lys Met Gln Lys 50 55 60 Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Asp 65 70 75 80 Gln Thr Ser Tyr Lys Leu Ile Asn Cys Gln Thr Ser Val Ile Thr Gln 85 90 95 Ala Cys Pro Lys 100

Claims

1. An expression cassette, comprising a polynucleotide sequence encoding a polypeptide including an HIV Gag polypeptide, wherein the polynucleotide sequence encoding said Gag polypeptide comprises a sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; and SEQ ID NO:16.

2-10. (canceled)

11. A recombinant expression system for use in a selected host cell, comprising, an expression cassette of claim 1, and wherein said polynucleotide sequence is operably linked to control elements compatible with expression in the selected host cell.

12. The recombinant expression system of claim 11, wherein said control elements are selected from the group consisting of a transcription promoter, a transcription enhancer element, a transcription termination signal, polyadenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences.

13. The recombinant expression system of claim 11, wherein said transcription promoter is selected from the group consisting of CMV, CMV+intron A, SV40, RSV, HIV-Ltr, MMLV-ltr, and metallothionein.

14. A cell comprising an expression cassette of claim 1, and wherein said polynucleotide sequence is operably linked to control elements compatible with expression in the selected cell.

15. The cell of claim 14, wherein the cell is a mammalian cell.

16. The cell of claim 15, wherein the cell is selected from the group consisting of BHK, VERO, HT1080, 293, RD, COS-7, and CHO cells.

17. The cell of claim 16, wherein said cell is a CHO cell.

18. The cell of claim 14, wherein the cell is an insect cell.

19. The cell of claim 18, wherein the cell is either Trichoplusia ni (Tn5) or Sf9 insect cells.

20. The cell of claim 14, wherein the cell is a bacterial cell.

21. The cell of claim 14, wherein the cell is a yeast cell.

22. The cell of claim 14, wherein the cell is a plant cell.

23. The cell of claim 14, wherein the cell is an antigen presenting cell.

24. The cell of claim 23, wherein the antigen presenting cell is a lymphoid cell selected from the group consisting of macrophages, monocytes, dendritic cells, B-cells, T-cells, stem cells, and progenitor cells thereof.

25. The cell of claim 14, wherein the cell is a primary cell.

26. The cell of claim 14, wherein the cell is an immortalized cell.

27. The cell of claim 14, wherein the cell is a tumor-derived cell.

28. (canceled)

29. A gene delivery vector for use in a mammalian subject, comprising a suitable gene delivery vector for use in said subject, wherein the vector comprises an expression cassette of claim 1, and wherein said polynucleotide sequence is operably linked to control elements compatible with expression in the subject.

30-57. (canceled)