CA2393861A1 - Polynucleotide vaccines expressing codon optimized hiv-1 nef and modified hiv-1 nef - Google Patents

Abstract

Pharmaceutical compositions which comprise HIV Nef DNA vaccines are disclosed, along with the production and use of these DNA vaccines. The nef-based DNA
vaccines of the invention are administered directly introduced into living vertebrate tissue, preferably humans, and express the HIV Nef protein or biologically relevant portions thereof, inducing a cellular immune response which specifically recognizes human immunodeficiency virus-1 (HIV-1). The DNA
molecules which comprise the open reading frame of these DNA vaccines are synthetic DNA molecules encoding codon optimized HIV-1 Nef and derivatives of optimized HIV-1 Nef, including nef modifications comprising amino terminal leader peptides, removal of the amino terminal myristylation site, and/or modification of the Nef dileucine motif. These modifications may effect wild type characteristics of Nef, such as myristylation and down regulation of host CD4.

Description

TITLE OF THE INVENTION

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit, under 35 U.S.C. ~119(e), of U.S.
provisional application 60/172,442, filed December 17, 1999.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable REFERENCE TO MICROFICHE APPENDIX
Not Applicable FIELD OF THE INVENTION
The present invention relates to HIV Nef polynucleotide pharmaceutical products, as well as the production and use thereof which, when directly introduced into living vertebrate tissue, preferably a mammalian host such as a human or a non-human mammal of commercial or domestic veterinary importance, express the HIV Nef protein or biologically relevant portions thereof within the animal, inducing a cellular immune response which specifically recognizes human immunodeficiency virus-1 (HIV-1). The polynucleotides of the present invention are synthetic DNA
molecules encoding codon optimized HIV-1 Nef and derivatives of optimized HIV-Nef, including nef mutants which effect wild type characteristics of Nef, such as myristylation and down regulation of host CD4. The polynucleotide vaccines of the present invention should offer a prophylactic advantage to previously uninfected individuals and/or provide a therapeutic effect by reducing viral load levels within an infected individual, thus prolonging the asymptomatic phase of HIV-1 infection.

BACKGROUND OF THE INVENTION
Human Immunodeficiency Virus-1 (HIV-1) is the etiological agent of acquired human immune deficiency syndrome (AIDS) and related disorders. HIV-1 is an RNA virus of the Retroviridae family and exhibits the 5' LTR-gag-pol-env-LTR 3' organization of all retroviruses. The integrated form of HIV-1, known as the provirus, is approximately 9.8 Kb in length. Each end of the viral genome contains flanking sequences known as long terminal repeats (LTRs). The HIV genes encode at least nine proteins and are divided into three classes; the major structural proteins (Gag, Pol, and Envy, the regulatory proteins (Tat and Rev); and the accessory proteins (Vpu, Vpr, Vif and Nef).
The gag gene encodes a 55-kilodalton (kDa) precursor protein (p55) which is expressed from the unspliced viral mRNA and is proteolytically processed by the HIV
protease, a product of the pol gene. The mature p55 protein products are p17 (matrix), p24 (capsid), p9 (nucleocapsid) and p6.
The pol gene encodes proteins necessary for virus replication; a reverse transcriptase, a protease, integrase and RNAse H. These viral proteins are expressed as a Gag-Pol fusion protein, a 160 kDa precursor protein which is generated via a ribosomal frame shifting. The viral encoded protease proteolytically cleaves the Pol polypeptide away from the Gag-Pol fusion and further cleaves the Pol polypeptide to the mature proteins which provide protease (Pro, P10), reverse transcriptase (RT, P50), integrase (IN, p31) and RNAse H (RNAse, p15) activities.
The nef gene encodes an early accessory HIV protein (Nef) which has been shown to possess several activities such as down regulating CD4 expression, disturbing T-cell activation and stimulating HIV infectivity.
The env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gp160) and then cleaved by a cellular protease to yield the external 120-kDa envelope glycoprotein (gp120) and the transmembrane kDa envelope glycoprotein (gp41). Gp120 and gp41 remain associated and are displayed on the viral particles and the surface of HIV-infected cells.
The tat gene encodes a long form and a short form of the Tat protein, a RNA
binding protein which is a transcriptional transactivator essential for HIV-1 replication.
The rev gene encodes the 13 kDa Rev protein, a RNA binding protein. The Rev protein binds to a region of the viral RNA termed the Rev response element (RRE). The Rev protein is promotes transfer of unspliced viral RNA from the nucleus to the cytoplasm. The Rev protein is required for HIV late gene expression and in turn, HIV replication.
Gp120 binds to the CD4/chemokine receptor present on the surface of helper T-lymphocytes, macrophages and other target cells in addition to other co-receptor molecules. X4 (macrophage tropic) virus show tropism for CD4/CXCR4 complexes while a RS (T-cell line tropic) virus interacts with a CD4/CCRS receptor complex.
After gp120 binds to CD4, gp41 mediates the fusion event responsible for virus entry.
The virus fuses with and enters the target cell, followed by reverse transcription of its single stranded RNA genome into the double-stranded DNA via a RNA dependent DNA polymerase. The viral DNA, known as provirus, enters the cell nucleus, where the viral DNA directs the production of new viral RNA within the nucleus, expression of early and late HIV viral proteins, and subsequently the production and cellular release of new virus particles. Recent advances in the ability to detect viral load within the host shows that the primary infection results in an extremely high generation and tissue distribution of the virus, followed by a steady state level of virus (albeit through a continual viral production and turnover during this phase), leading ultimately to another burst of virus load which leads to the onset of clinical AIDS.
Productively infected cells have a half life of several days, whereas chronically or latently infected cells have a 3-week half life, followed by non-productively infected cells which have a long half life (over 100 days) but do not significantly contribute to day to day viral loads seen throughout the course of disease.
Destruction of CD4 helper T lymphocytes, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of HIV infection. The loss of CD4 T-cells seriously impairs the body's ability to fight most invaders, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.
Effective treatment regimens for HIV-1 infected individuals have become available recently. However, these drugs will not have a significant impact on the disease in many parts of the world and they will have a minimal impact in halting the spread of infection within the human population. As is true of many other infectious diseases, a significant epidemiologic impact on the spread of HIV-1 infection will only occur subsequent to the development and introduction of an effective vaccine.
There are a number of factors that have contributed to the lack of successful vaccine development to date. As noted above, it is now apparent that in a chronically infected person there exists constant virus production in spite of the presence of anti-humoral and cellular immune responses and destruction of virally infected cells. As in the case of other infectious diseases, the outcome of disease is the result of a balance between the kinetics and the magnitude of the immune response and the pathogen replicative rate and accessibility to the immune response. Pre-existing immunity may be more successful with an acute infection than an evolving immune response can be with an established infection. A second factor is the considerable genetic variability of the virus. Although anti-HIV-1 antibodies exist that can neutralize HIV-1 infectivity in cell culture, these antibodies are generally virus isolate-specific in their activity. It has proven impossible to define serological groupings of HIV-1 using traditional methods. Rather, the virus seems to define a serological "continuum" so that individual neutralizing antibody responses, at best, are effective against only a handful of viral variants. Given this latter observation, it would be useful to identify immunogens and related delivery technologies that are likely to elicit anti-HIV-1 cellular immune responses. It is known that in order to generate CTL responses antigen must be synthesized within or introduced into cells, subsequently processed into small peptides by the proteasome complex, and translocated into the endoplasmic reticulum/Golgi complex secretory pathway for eventual association with major histocompatibility complex (MHC) class I
proteins.
CD8+ T lymphocytes recognize antigen in association with class I MHC via the T
cell receptor (TCR) and the CD8 cell surface protein. Activation of naive CD8+ T
cells into activated effector or memory cells generally requires both TCR engagement of antigen as described above as well as engagement of costimulatory proteins.
Optimal induction of CTL responses usually requires "help" in the form of cytokines from CD4+ T lymphocytes which recognize antigen associated with MHC class II
molecules via TCR and CD4 engagement.
As introduced above, the nef gene encodes an early accessory HIV protein (Nef) which has been shown to possess several activities such as down regulating CD4 expression, disturbing T-cell activation and stimulating HIV infectivity.
Zazopoulos and Haseltine (1992, Proc. Natl. Acad. Sci. 89: 6634-6638) disclose mutations to the HIV-1 nef gene which effect the rate of virus replication.
The authors show that the nef open reading frame mutated to encode Ala-2 in place of Gly-2 inhibits myristolation of the protein and results in delayed viral replication rates in Jurkat cells and PBMCs.
Kaminchik et al. (1991, J. Virology 65(2): 583-588) disclose an amino-terminal nef open reading frame mutated to encode Met-Ala-Ala in place of Met-Gly-Gly. The authors show that this mutant is deficient in myristolation.
Saksela et al. (1995, EMBO J. 14(3): 484-491) and Lee et al. (1995, EMBO J.
14(20): 5006-5015) show the importance of a proline rich motif in HIV-1 Nef which mediates binding to a SH3 domain of the Hck protein. The authors conclude that this motif is important in the enhancement of viral replication but not down-regulation of CD4 expression.
Calarota et al. (1998, The Lancet 351: 1320-1325) present human clinical data concerning immunization of three HIV infected individuals with a DNA plasmid expressing wild type Nef. The authors conclude that immunization with a Nef encoding DNA plasmid induced a cellular immune response in the three individuals.
However, two of the three patients were on alternative therapies during the study, and the authors conclude that the CTL response was most likely a boost to a pre-existing CTL response. In addition, the viral load increased substantially in two of the three patients during the course of the study.
Tobery et al. (1997, J. Exp. Med. 185(5): 909-920) constructed two ubiquitin nef (Ub-nef) fusion constructs, one which encoded the Nef initiating methionine and the other with an Arg residue at the amino terminus of the Nef open reading frame.
The authors state that vaccinia- or plasmid-based immunization of mice with a Ub-nef construct containing an Arg residue at the amino terminus induces a Nef-specific CTL
response. The authors suggest the expressed fusion protein is more efficiently presented to the MHC class I antigen presentation pathway, resulting in an improved cellular immune response.
Kim et al. (1997, J. Immunol. 158(2): 816-826) disclose that co-administration of a plasmid DNA construct expressing IL-12 with a plasmid construct expressing Nef results in an improved cellular immune response in mice when compared to inoculation with the Nef construct alone. The authors reported a reduction in the humoral response from the Nef / IL-12 co-administration as compared to administration of the plasmid construct expressing Nef alone.
Moynier et al. (1998, Vaccine 16(16): 1523-1530) show varying humoral responses in mice immunized with a DNA plasmid encoding Nef, depending upon the presence of absence of Freund's adjuvant. No data is disclosed regarding a cellular immune response in mice vaccinated with the aforementioned DNA construct alone.
Hanna et al. (1998, Cell 95:163-175) suggest that wild type Nef may play a critical role in AIDS pathogenicity.
It would be of great import in the battle against A>DS to produce a prophylactic- and/or therapeutic-based HIV vaccine which generates a strong cellular immune response against an HIV infection. The present invention addresses and meets this needs by disclosing a class of DNA vaccines based on host delivery and expression of the early HIV gene, nef.
SUMMARY OF THE INVENTION
The present invention relates to synthetic DNA molecules (also referred to herein as "polynucleotides") and associated DNA vaccines (also referred to herein as "polynucleotide vaccines") which elicit CTL responses upon administration to the host, such as a mammalian host and including primates and especially humans, as well as non-human mammals of commercial or domestic veterinary importance.
The CTL-directed vaccines of the present invention should lower transmission rate to previously uninfected individuals and/or reduce levels of the viral loads within an infected individual, so as to prolong the asymptomatic phase of HIV-1 infection. In particular, the present invention relates to DNA vaccines which encode various forms of HIV-1 Nef, wherein administration, intracellular delivery and expression of the HIV-1 nef gene of interest elicits a host CTL and Th response. The preferred synthetic DNA molecules of the present invention encode codon optimized versions of wild type HIV-1 Nef, codon optimized versions of HIV-1 Nef fusion proteins, and codon optimized versions of HIV-1 Nef derivatives, including but not limited to nef modifications involving introduction of an amino-terminal leader sequence, removal of an amino-terminal myristylation site and/or introduction of dileucine motif mutations. The Nef-based fusion and modified proteins disclosed within this specification may possess altered trafficking and/or host cell function while retaining the ability to be properly presented to the host MHC I complex and in turn elicit a host CTL and Th response.
A particular embodiment of the present invention relates to a DNA molecule encoding HIV-1 Nef from the HIV-1 jfrl isolate wherein the codons are optimized for expression in a mammalian system such as a human. The DNA molecule which encodes this protein is disclosed herein as SEQ ID NO:1, while the expressed open reading frame is disclosed herein as SEQ ID N0:2.
In another embodiment of the present invention, a codon optimized DNA
molecule encoding a protein containing the human plasminogen activator (tpa) leader peptide fused with the NHz-terminus of the HIV-1 Nef polypeptide. The DNA
molecule which encodes this protein is disclosed herein as SEQ ID N0:3, while the expressed open reading frame is disclosed herein as SEQ 117 N0:4.
In an additional embodiment, the present invention relates to a DNA molecule encoding optimized HIV-1 Nef wherein the open reading frame codes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175, herein described as opt nef (G2A,LLAA). The DNA molecule which encodes this protein is disclosed herein as SEQ ID NO:S, while the expressed open reading frame is disclosed herein as SEQ B7 N0:6.
Another additional embodiment of the present invention relates to a DNA
molecule encoding optimized HIV-1 Nef wherein the amino terminal myristylation site and dileucine motif have been deleted, as well as comprising a tPA leader peptide.
This DNA molecule, opt tpanef (LLAA), comprises an open reading frame which encodes a Nef protein containing a tPA leader sequence fused to amino acid residue 6-216 of HIV-1 Nef (jfrl), wherein Leu-174 and Leu-175 are substituted with Ala-174 and Ala-175, herein referred to as opt tpanef (LLAA) is disclosed herein as SEQ >D
N0:7, while the expressed open reading frame is disclosed herein as SEQ ID
N0:8.
The present invention also relates to non-codon optimized versions of DNA
molecules and associated DNA vaccines which encode the various wild type and modified forms of the HIV Nef protein disclosed herein. Partial or fully codon optimized DNA vaccine expression vector constructs are preferred, but it is within the scope of the present invention to utilize "non-codon optimized" versions of the constructs disclosed herein, especially modified versions of HIV Nef which are shown to promote a substantial cellular immune response subsequent to host administration.
The DNA backbone of the DNA vaccines of the present invention are preferably DNA plasmid expression vectors. DNA plasmid expression vectors utilized in the present invention include but are not limited to constructs which comprise the cytomegalovirus promoter with the intron A sequence (CMV-intA) and a bovine growth hormone transcription termination sequence. In addition, the DNA
plasmid vectors of the present invention preferably comprise an antibiotic resistance _7_ marker, including but not limited to an ampicillin resistance gene, a neomycin resistance gene or any other pharmaceutically acceptable antibiotic resistance marker.
In addition, an appropriate polylinker cloning site and a prokaryotic origin of replication sequence are also preferred. Specific DNA vectors of the present invention include but are not limited to V1, V1J (SEQ 1D N0:14), VlJneo (SEQ
>D
NO:15), VlJns (Figure 1A, SEQ 117 N0:16), V1R (SEQ ID N0:26), and any of the aforementioned vectors wherein a nucleotide sequence encoding a leader peptide, preferably the human tPA leader, is fused directly downstream of the CMV-intA
promoter, including but not limited to VlJns-tpa, as shown in Figure 1B and SEQ ID
N0:19.
The present invention especially relates to a DNA vaccine and a pharmaceutically active vaccine composition which contains this DNA vaccine, and the use as a prophylactic and/or therapeutic vaccine for host immunization, preferably human host immunization, against an HIV infection or to combat an existing HIV
condition. These DNA vaccines are represented by codon optimized DNA molecules encoding HIV-1 Nef of biologically active Nef modifications or Nef-containing fusion proteins which are ligated within an appropriate DNA plasmid vector, with or without a nucleotide sequence encoding a functional leader peptide. DNA
vaccines of the present invention relate in part to codon optimized DNA molecules encoding HIV-1 Nef of biologically active Nef modifications or Nef-containing fusion proteins ligated in DNA vectors V 1, V 1J (SEQ ID N0:14), V lJneo (SEQ ID NO:15), V
lJns (Figure 1A, SEQ ID N0:16), V1R (SEQ ID N0:26), or any of the aforementioned vectors wherein a nucleotide sequence encoding a leader peptide, preferably the human tPA leader, is fused directly downstream of the CMV-intA promoter, including but not limited to VlJns-tpa, as shown in Figure 1B and SEQ >D
N0:19.
Especially preferred DNA vaccines of the present invention include codon optimized DNA vaccine constructs VlJns/nef, VlJns/tpanef, VlJns/tpanef(LLAA) and VlJns/(G2A,LLAA), as exemplified in Example Section 2.
The present invention also relates to HIV Nef polynucleotide pharmaceutical products, as well as the production and use thereof, wherein the DNA vaccines are formulated with an adjuvant or adjuvants which may increase immunogenicity of the DNA polynucleotide vaccines of the present invention, namely by increasing a humoral response to inoculation. A preferred adjuvant is an aluminum phosphate-based adjuvant or a calcium phosphate based adjuvant, _g-with an aluminum phosphate adjuvant being especially preferred. Another preferred adjuvant is a non-ionic block copolymer, preferably comprising the blocks of polyoxyethylene (POE) and polyoxypropylene (POP) such as a POE-POP-POE block copolymer. These adjuvanted forms comprising the DNA
vaccines disclosed herein are useful in increasing humoral responses to DNA
vaccination without imparting a negative effect on an appropriate cellular immune response.
As used herein, a DNA vaccine or DNA polynucleotide vaccine or polynucleotide vaccine is a DNA molecule (i.e., "nucleic acid", "polynucleotide") which contains essential regulatory elements such that upon introduction into a living, vertebrate cell, it is able to direct the cellular machinery to produce translation products encoded by the respective nef genes of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure lA-B show a schematic representation of DNA vaccine expression vectors VlJns (A) and VlJns/tpa utilized for HIV-1 nef and HIV-1 modified nef constructs.
Figure 2A-B show a nucleotide sequence comparison between wild type nef(jrfl) and codon optimized nef. The wild type nef gene from the jrfl isolate consists of 648 nucleotides capable of encoding a 216 amino acid polypeptide.
WT, wild type sequence (SEQ >D N0:9); opt, codon-optimized sequence (contained within SEQ m NO:1). The Nef amino acid sequence is shown in one-letter code (SEQ m N0:2).
Figure 3A-C show nucleotide sequences at junctions between nef coding sequence and plasmid backbone of nef expression vectors V lJns/nef (Figure 3A), VlJns/nef(G2A,LLAA) (Figure 3B), VlJns/tpanef (Figure 3C) and V lJns/tpanef(LLAA) (Figure 3C, also). 5' and 3' flanking sequences of codon optimized nef or codon optimized nef mutant genes are indicated by bold/italic letters;
nef and nef mutant coding sequences are indicated by plain letters. Also indicated (as underlined) are the restriction endonuclease sites involved in construction of respective nef expression vectors. VlJns/tpanef and VlJns/tpanef(LLAA) have identical sequences at the junctions.
Figure 4 shows a schematic presentation of nef and nef derivatives. Amino acid residues involved in Nef derivatives are presented. Glycine 2 and Leucine174 and 175 are the sites involved in myristylation and dileucine motif, respectively. For both versions of the tpanef fusion genes, the putative leader peptide cleavage sites are indicated with "*", and a exogenous serine residue introduced during the construction of the mutants is underlined.
Figure 5 shows Western blot analysis of nef and modified nef proteins expressed in transfected 293 cells. 293 cells grown in 100 mm culture dish were transfected with respective codon optimized nef constructs. Sixty hours post transfection, supernatant and cells were collected separately and separated on 10%
SDS-PAGE under reducing conditions. The proteins were transferred into a PVDF
membrane and probed with a mixture of Gag mAb and Nef mAbs, both at 1:2000 dilution. The protein signals were detected with ECL. (A) cells transfected with VlJns/gag only; (B) cells transfected with VlJns/gag and VlJns/nef; (C) cells transfected with VlJns/gag and VlJns/nef(G2A, LLAA); (D) cells transfected with V lJns/gag and V lJns/tpanef; (E) cells transfected with V lJns/gag and VlJns/tpanef(LLAA). The low case letter c and m represent medium and cellular fractions, respectively. M.W. = molecular weight marker.
Figure 6 shows an Elispot assay of cell-mediated responses to Nef peptides.
Three strains of mice, Balb/c, C57BIJ6 and C3H, were immunized with 50 mcg of V lJns/nef (codon optimized) and boosted twice with a two-week interval. Two weeks following the final immunization, splenocytes were isolated and tested in an Elispot assay against respective Nef peptide pools. As a control, splenocytes were from non-immunized naive mice were tested in parallel. Nef peptide pool A
consists of all 21 Nef peptides; Nef peptide pool B consists of 11 non-overlapping peptide started from residue 1; Nef peptide pool C consists of 10 non-overlapping peptides started from residue 11. SFC, INF-gamma secreting spot-forming cells.
Figure 7A-C show Nef-specific CD8 and CD4 epitope mapping. The immunization regime is as per Figure 6. Mouse splenocytes were isolated and fractionated into CD8+ and CD8- cells using Miltenyi's magnetic cell separator. The resultant CD8+ and CD8- cells were then tested in an Elispot assay against individual Nef peptides. SFC, INF-gamma secreting spot-forming cells. The mice strains tested are Balb/c mice (Figure 7A), C57BL/6 mice (Figure 7B), and C3H mice (Figure 7C).
Figure 8A-C show identification of a Nef CTL epitope. Splenocytes from nef immunized C57BL/6 mice were stimulated in vitro with peptide-pulsed, irradiated naiva splenocytes for 7 days. Following the in vitro stimulation, cells were harvested and tested in a standard S~Cr-releasing assay using peptide pulsed EL-4 cells as targets. Open symbol, specific killings of EL-4 cells without peptide; solid symbol, specific killing of EL-4 cells with peptide. Panel A - peptide Nef 51-70;
Panel B -peptide Nef 60-68, Panel C - peptide Nef 58-70.
Figure 9A-B shows a comparison of the immunogenicity of codon optimized DNA vaccine vectors expressing Nef and modified forms of Nef C57BL/6 mice, five per group, were immunized with 100 mcg of the indicated nef constructs.
Fourteen days following immunization, splenocytes were collected and tested against the Nef CD8 (aa58-66) and CD4 (aa81-100) peptides. Identical immunization regimens were used for both experiments. In experiment 1 (Panel A), three codon optimized nef constructs were tested, namely, VlJns/nef, VlJns/tpanef(LLAA) and VlJns/nef(G2A,LLAA), whereas in experiment 2 (Panel B) all four codon optimized nef constructs were tested. The data represent means plus standard deviation of 5 mice per group.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to synthetic DNA molecules (also referred to herein as "nucleic acid" molecules or "polynucleotides") and associated DNA
vector vaccines (also referred to herein as "polynucleotide vaccines") which elicit CTL and humoral responses upon administration to the host, including primates and especially humans. In particular, the present invention relates to DNA vector vaccines which encode various forms of HIV-1 Nef, wherein administration, intracellular delivery and expression of the HIV-1 nef gene of interest elicits a host CTL and Th response.
The synthetic DNA molecules of the present invention encode codon optimized versions of wild type HIV-1 Nef, codon optimized versions of HIV-1 Nef fusion proteins, and codon optimized versions of HIV-1 Nef derivatives, including but not limited to nef modifications involving introduction of an amino-terminal leader sequence, removal of an amino-terminal myristylation site and/or introduction of dileucine motif mutations. In some instances the Nef-based fusion and modified proteins disclosed within this specification possess altered trafficking and/or host cell function while retaining the ability to be properly presented to the host MHC
I
complex. Those skilled in the art will recognize that the use of nef genes from HIV-2 strains which express Nef proteins having analogous function to HIV-1 Nef would be expected to generate immune responses analogous to those described herein for HIV-1 constructs.
In order to generate a CTL response, the immunogen must be synthesized within (MHCI presentation) or introduced into cells (MHCII presentation). For intracellular synthesized immunogens, the protein is expressed and then processed into small peptides by the proteasome complex, and translocated into the endoplasmic reticulum/Golgi complex secretory pathway for eventual association with major histocompatibility complex (MHC) class I proteins. CD8+ T lymphocytes recognize antigen in association with class I MHC via the T cell receptor (TCR).
Activation of naive CD8+ T cells into activated effector or memory cells generally requires both TCR engagement of antigen as described above as well as engagement of co-stimulatory proteins. Optimal induction of CTL responses usually requires "help"
in the form of cytokines from CD4+ T lymphocytes which recognize antigen associated with MHC class II molecules via TCR.
The HIV-1 genome employs predominantly uncommon codons compared to highly expressed human genes. Therefore, the nef open reading frame has been synthetically manipulated using optimal codons for human expression. As noted above, a preferred embodiment of the present invention relates to DNA
molecules which comprise a HIV-1 nef open reading frame, whether encoding full length nef or a modification or fusion as described herein, wherein the codon usage has been optimized for expression in a mammal, especially a human.
In a particular embodiment of the present invention, a DNA molecule encoding HIV-1 Nef from the HIV-1 jfrl isolate wherein the codons are optimized for expression in a mammalian system such as a human. The nucleotide sequence of the codon optimized version of HIV-1 jrfl nef gene is disclosed herein as SEQ ID
NO:1, as shown herein:
GATCTGCCAC CATGGGCGGC AAGTGGTCCA AGAGGTCCGT GCCCGGCTGG TCCACCGTGA
GGGAGAGGAT GAGGAGGGCC GAGCCCGCCG CCGACAGGGT GAGGAGGACC GAGCCCGCCG
CCGTGGGCGT GGGCGCCGTG TCCAGGGACC TGGAGAAGCA CGGCGCCATC ACCTCCTCCA
ACACCGCCGC CACCAACGCC GACTGCGCCT GGCTGGAGGC CCAGGAGGAC GAGGAGGTGG

TGTCCCACTT CCTGAAGGAG AAGGGCGGCC TGGAGGGCCT GATCCACTCC CAGAAGAGGC
AGGACATCCT GGACCTGTGG GTGTACCACA CCCAGGGCTA CTTCCCCGAC TGGCAGAACT
ACACCCCCGG CCCCGGCATC AGGTTCCCCC TGACCTTCGG CTGGTGCTTC AAGCTGGTGC
CCGTGGAGCC CGAGAAGGTG GAGGAGGCCA ACGAGGGCGA GAACAACTGC CTGCTGCACC

CCATGTCCCA GCACGGCATC GAGGACCCCG AGAAGGAGGT GCTGGAGTGG AGGTTCGACT
CCAAGCTGGC CTTCCACCAC GTGGCCAGGG AGCTGCACCC CGAGTACTAC AAGGACTGCT
AAAGCCCGGG C (SEQ ID N0:1).
As can be discerned from comparing native to optimized codon usage in Figure 2A-B, the following codon usage for mammalian optimization is preferred:
Met (ATG), Gly (GGC), Lys (AAG), Trp (TGG), Ser (TCC), Arg (AGG), Val (GTG), Pro (CCC), Thr (ACC), Glu (GAG); Leu (CTG), His (CAC), Ile (ATC), Asn (AAC), Cys (TGC), Ala (GCC), Gln (CAG), Phe (TTC) and Tyr (TAC). For an additional discussion relating to mammalian (human) codon optimization, see WO 97/31115 (PCT/US97/02294), which is hereby incorporated by reference.
The open reading frame for SEQ ID NO:l above comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662. The open reading frame of SEQ ID NO:1 provides for a 216 amino acid HIV-1 Nef protein expressed through utilization of a codon optimized DNA
vaccine vector. The 216 amino acid HIV-1 Nef (jfrl) protein is disclosed herein as SEQ
ID
N0:2, and as follows:
Met Gly Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys (SEQ ID N0:2).
HIV-1 Nef is a 206 amino acid cytosolic protein which associates with the inner surface of the host cell plasma membrane through myristylation of Gly-2 (Franchini et al., 1986, Virology 155: 593-599). While not all possible Nef functions have been elucidated, it has become clear that correct trafficking of Nef to the inner plasma membrane promotes viral replication by altering the host intracellular environment to facilitate the early phase of the HIV-1 life cycle and by increasing the infectivity of progeny viral particles. In one aspect of the invention regarding codon-optimized, protein-modified polypeptides, either the DNA vaccine vector molecule or the HIV-1 nef construct is modified to contain a nucleotide sequence which encodes a heterologous leader peptide such that the amino terminal region of the expressed protein will contain the leader peptide. The diversity of function that typifies eukaryotic cells depends upon the structural differentiation of their membrane boundaries. To generate and maintain these structures, proteins must be transported from their site of synthesis in the endoplasmic reticulum to predetermined destinations throughout the cell. This requires that the trafficking proteins display sorting signals that are recognized by the molecular machinery responsible for route selection located at the access points to the main trafficking pathways.
Sorting decisions for most proteins need to be made only once as they traverse their biosynthetic pathways since their final destination, the cellular location at which they perform their function, becomes their permanent residence. Maintenance of intracellular integrity depends in part on the selective sorting and accurate transport of proteins to their correct destinations. Defined sequence motifs exist in proteins which can act as 'address labels'. A number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins. An effective induction of CTL
responses often required sustained, high level endogenous expression of an antigen. In light of its diverse biological activities, vaccines composed of wild-type Nef could potentially have adverse effects on the host cells. As membrane-association via myristylation is an essential requirement for most of Nef's function, mutants lacking myristylation, by glycine-to-alanine change, change of the dileucine motif andJor by substitution with a tpa leader sequence as described herein, will be functionally defective, and therefore will have improved safety profile compared to wild-type Nef for use as an HIV-1 vaccine component.
In a preferred and exemplified embodiment of this portion of the invention, either the DNA vector or the HIV-1 nef nucleotide sequence is modified to include the human tissue-specific plasminogen activator (tPA) leader. As shown in Figure lA-B for the DNA vector VlJns, a DNA vector which may be utilized to practice the present invention may be modified by known recombinant DNA
methodology to contain a leader signal peptide of interest, such that downstream cloning of the modified HIV-1 protein of interest results in a nucleotide sequence which encodes a modified HIV-1 tPA/Nef protein. In the alternative, as noted above, insertion of a nucleotide sequence which encodes a leader peptide may be inserted into a DNA vector housing the open reading frame for the Nef protein of interest.
Regardless of the cloning strategy, the end result is a polynucleotide vaccine which comprises vector components for effective gene expression in conjunction with nucleotide sequences which encode a modified HIV-1 Nef protein of interest, including but not limited to a HIV-1 Nef protein which contains a leader peptide. The amino acid sequence of the human tPA leader utilized herein is as follows:
MDAMKRGLCCVLLLCGAVFVSPSEISS (SEQ m N0:19).
It has been shown that myristylation of Gly-2 in conjunction with a dileucine motif in the carboxy region of the protein is essential for Nef-induced down regulation of CD4 (Aiken et al., 1994, Cell 76: 853-864) via endocytosis. It has also been shown that Nef expression promotes down regulation of MHCI (Schwartz et al., 1996, Nature Medicine 2(3): 338-342) via endocytosis. The present invention relates in part to DNA vaccines which encode modified Nef proteins altered in trafficking and/or functional properties. The modifications introduced into the DNA
vaccines of the present invention include but are not limited to additions, deletions or substitutions to the nef open reading frame which results in the expression of a modified Nef protein which includes an amino terminal leader peptide, modification or deletion of the amino terminal myristylation site, and modification or deletion of the dileucine motif within the Nef protein and which alter function within the infected host cell. Therefore, a central theme of the DNA molecules and DNA vaccines of the present invention is (1) host administration and intracellular delivery of a codon optimized nef-based DNA vector vaccine; (2) expression of a modified Nef protein which is immunogenic in terms of eliciting both CTL and Th responses; and, (3) inhibiting or at least altering known early viral functions of Nef which have been shown to promote HIV-1 replication and load within an infected host.
In another preferred and exemplified embodiment of the present invention, the nef coding region is altered, resulting in a DNA vaccine which expresses a modified Nef protein wherein the amino terminal Gly-2 myristylation residue is either deleted or modified to express alternate amino acid residues.
In another preferred and exemplified embodiment of the present invention, the nef coding region is altered, resulting in a DNA vaccine which expresses a modified Nef protein wherein the dileucine motif is either deleted or modified to express alternate amino acid residues.
Therefore, the present invention relates to an isolated DNA molecule, regardless of codon usage, which expresses a wild type or modified Nef protein as described herein, including but not limited to modified Nef proteins which comprise a deletion or substitution of Gly 2, a deletion or substitution of Leu 174 and Leu 17S
and/or inclusion of a leader sequence.
The present invention also relates to a substantially purified protein expressed from the DNA polynucleotide vaccines of the present invention, especially the purified proteins set forth below as SEQ ID NOs: 2, 4, 6, and 8. These purified proteins may be useful as protein-based HIV vaccines.
In a specific embodiment of the invention as it relates DNA vaccines encoding modified forms of HIV-1, an open reading frame which encodes a Nef protein which comprises a tPA leader sequence fused to amino acid residue 6-216 of HIV-1 Nef 1S (jfrl) is referred to herein as opt tpanef. The nucleotide sequence comprising the open reading frame of opt tpanef is disclosed herein as SEQ ID N0:3, as shown below:
CATGGATGCA ATGAAGAGAG GGCTCTGCTG TGTGCTGCTG CTGTGTGGAG CAGTCTTCGT
TTCGCCCAGC GAGATCTCCT CCAAGAGGTC CGTGCCCGGC TGGTCCACCG TGAGGGAGAG
GATGAGGAGG GCCGAGCCCG CCGCCGACAG GGTGAGGAGG ACCGAGCCCG CCGCCGTGGG

CGCCACCAAC GCCGACTGCG CCTGGCTGGA GGCCCAGGAG GACGAGGAGG TGGGCTTCCC
CGTGAGGCCC CAGGTGCCCC TGAGGCCCAT GACCTACAAG GGCGCCGTGG ACCTGTCCCA
CTTCCTGAAG GAGAAGGGCG GCCTGGAGGG CCTGATCCAC TCCCAGAAGA GGCAGGACAT
CCTGGACCTG TGGGTGTACC ACACCCAGGG CTACTTCCCC GACTGGCAGA ACTACACCCC
ZS CGGCCCCGGC ATCAGGTTCC CCCTGACCTT CGGCTGGTGC TTCAAGCTGG TGCCCGTGGA
GCCCGAGAAG GTGGAGGAGG CCAACGAGGG CGAGAACAAC TGCCTGCTGC ACCCCATGTC
CCAGCACGGC ATCGAGGACC CCGAGAAGGA GGTGCTGGAG TGGAGGTTCG ACTCCAAGCT
GGCCTTCCAC CACGTGGCCA GGGAGCTGCA CCCCGAGTAC TACAAGGACT GCTAAAGCC
(SEQ ID N0:3).
30 The open reading frame for SEQ ID N0:3 comprises an initiating methionine residue at nucleotides4 and a stop 2- "TAA" codon from nucleotides 713-715.
The open reading frameEQ ID N0:3 Nef of S provides for a 237 amino acid HIV-1 protein which comprises nce fused 6-216 a tPA leader seque to amino of acids HIV-1 Nef, including acid residues the dileucine 174 and motif at amino 175. This amino acid tPA/Nef as SEQ :4, (jfrl) fusion ID and protein is disclosed N0 is herein shown as follows:

Met Asp Ala Met Arg Gly CysCys Val Leu Cys Gly Lys Leu Leu Leu Ala Val Phe Val Pro Ser IleSer Ser Arg Val Pro Ser Glu Lys Ser Gly Trp Ser Thr Arg Glu MetArg Arg Glu Ala Ala Val Arg Ala Pro Asp Arg Val Arg Thr Glu AlaAla Val Val Ala Val Arg Pro Gly Gly Ser Arg Asp Leu Lys His AlaIle Thr Ser Thr Ala Glu Gly Ser Asn Ala Thr Asn Ala Cys Ala LeuGlu Ala Glu Glu Glu Asp Trp Gln Asp Val Gly Phe Pro Arg Pro ValPro Leu Pro Thr Tyr Val Gln Arg Met Lys Gly Ala Val Leu Ser PheLeu Lys Lys Gly Leu Asp His Glu Gly Glu Gly Leu Ile Ser Gln ArgGln Asp Leu Leu Trp His Lys Ile Asp Val Tyr His Thr Gly Tyr ProAsp Trp Asn Thr Pro Gln Phe Gln Tyr Gly Pro Gly Ile Phe Pro ThrPhe Gly Cys Lys Leu Arg Leu Trp Phe Val Pro Val Glu Glu Lys GluGlu Ala Glu Glu Asn Pro Val Asn Gly Asn Cys Leu Leu Pro Met GlnHis Gly Glu Pro Glu His Ser Ile Asp Lys Glu Val Leu Trp Arg AspSer Lys Ala His His Glu Phe Leu Phe Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys (SEQ ID N0:4).
Therefore, this exemplified Nef protein, Opt tPA-Nef, contains both a tPA
leader sequence as well as deleting the myristylation site of Gly-2A DNA
molecule encoding HIV-1 Nef from the HIV-1 jfrl isolate wherein the codons are optimized for expression in a mammalian system such as a human.
In another specific embodiment of the present invention, a DNA molecule is disclosed which encodes optimized HIV-1 Nef wherein the open reading frame codes for modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175. This open reading frame is herein described as opt nef (G2A,LLAA) and is disclosed as SEQ ID
NO:S, which comprises an initiating methionine residue at nucleotides 12-14 and a "TAA" stop codon from nucleotides 660-662. The nucleotide sequence of this codon optimized version of HIV-1 jrfl nef gene with the above mentioned modifications is disclosed herein as SEQ ID NO:S, as follows:

GATCTGCCAC CATGGCCGGC AAGTGGTCCA AGAGGTCCGTGCCCGGCTGGTCCACCGTGA

GGGAGAGGAT GAGGAGGGCC GAGCCCGCCG CCGACAGGGTGAGGAGGACCGAGCCCGCCG

CCGTGGGCGT GGGCGCCGTG TCCAGGGACC TGGAGAAGCACGGCGCCATCACCTCCTCCA

ACACCGCCGC CACCAACGCC GACTGCGCCT GGCTGGAGGCCCAGGAGGACGAGGAGGTGG

S GCTTCCCCGT GAGGCCCCAG GTGCCCCTGA GGCCCATGACCTACAAGGGCGCCGTGGACC

TGTCCCACTT CCTGAAGGAG AAGGGCGGCC TGGAGGGCCTGATCCACTCCCAGAAGAGGC

AGGACATCCT GGACCTGTGG GTGTACCACA CCCAGGGCTACTTCCCCGACTGGCAGAACT

ACACCCCCGG CCCCGGCATC AGGTTCCCCC TGACCTTCGGCTGGTGCTTCAAGCTGGTGC

CCGTGGAGCC CGAGAAGGTG GAGGAGGCCA ACGAGGGCGAGAACAACTGCGCCGCCCACC

CCATGTCCCA GCACGGCATC GAGGACCCCG AGAAGGAGGTGCTGGAGTGGAGGTTCGACT

CCAAGCTGGC CTTCCACCAC GTGGCCAGGG AGCTGCACCCCGAGTACTACAAGGACTGCT

AAAGCCCGGG C (SEQ ID N0:5).

The open reading frame of SEQ m NO:S ,LLAA), encodes Nef (G2A

disclosed herein as SEQ ID N0:6, as follows:

1S Met Ala Gly Lys Trp Ser Lys Arg Ser Gly Trp Thr Val Val Pro Ser Arg Glu Arg Met Arg Arg Ala Glu Pro Asp Arg Arg Arg Ala Ala Val Thr Glu Pro Ala Ala Val Gly Val Gly Ser Arg Leu Glu Ala Val Asp Lys His Gly Ala Ile Thr Ser Ser Asn Ala Thr Ala Asp Thr Ala Asn Cys Ala Trp Leu Glu Ala Gln Glu Asp Val Gly Pro Val Glu Glu Phe Arg Pro Gln Val Pro Leu Arg Pro Met Lys Gly Val Asp Thr Tyr Ala Leu Ser His Phe Leu Lys Glu Lys Gly Glu Gly Ile His Gly Leu Leu Ser Gln Lys Arg Gln Asp Ile Leu Asp Val Tyr Thr Gln Leu Trp His Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Gly Pro Ile Arg Thr Pro Gly Phe Pro Leu Thr Phe Gly Trp Cys Phe Val Pro Glu Pro Lys Leu Val 2S Glu Lys Val Glu Glu Ala Asn Glu Gly Asn Cys Ala His Glu Asn Ala Pro Met Ser Gln His Gly Ile Glu Asp Lys Glu Leu Glu Pro Glu Val Trp Arg Phe Asp Ser Lys Leu Ala Phe Val Ala Glu Leu His His Arg His Pro Glu Tyr Tyr Lys Asp Cys Ser (SEQ ID N0:6).
An additional embodiment of the present invention relates to another DNA
molecule encoding optimized HIV-1 Nef wherein the amino terminal myristylation site and dileucine motif have been deleted, as well as comprising a tPA leader peptide.
This DNA molecule, opt tpanef (LLAA) comprises an open reading frame which encodes a Nef protein containing a tPA leader sequence fused to amino acid residue 6-216 of HIV-1 Nef (jfrl), wherein Leu-174 and Leu-17S are substituted with Ala-174 and Ala-17S (Ala-19S and Ala-196 in this tPA-based fusion protein). The nucleotide sequence comprising the open reading frame of opt tpanef (LLAA) is disclosed herein as SEQ >D N0:7, as shown below:

CATGGATGCA ATGAAGAGAG GGCTCTGCTG TGTGCTGCTGCTGTGTGGAG CAGTCTTCGT

S TTCGCCCAGC GAGATCTCCT CCAAGAGGTC CGTGCCCGGCTGGTCCACCG TGAGGGAGAG

GATGAGGAGG GCCGAGCCCG CCGCCGACAG GGTGAGGAGGACCGAGCCCG CCGCCGTGGG

CGTGGGCGCC GTGTCCAGGG ACCTGGAGAA GCACGGCGCCATCACCTCCT CCAACACCGC

CGCCACCAAC GCCGACTGCG CCTGGCTGGA GGCCCAGGAGGACGAGGAGG TGGGCTTCCC

CGTGAGGCCC CAGGTGCCCC TGAGGCCCAT GACCTACAAGGGCGCCGTGG ACCTGTCCCA

IO CTTCCTGAAG GAGAAGGGCG GCCTGGAGGG CCTGATCCACTCCCAGAAGA GGCAGGACAT

CCTGGACCTG TGGGTGTACC ACACCCAGGG CTACTTCCCCGACTGGCAGA ACTACACCCC

CGGCCCCGGC ATCAGGTTCC CCCTGACCTT CGGCTGGTGCTTCAAGCTGG TGCCCGTGGA

GCCCGAGAAG GTGGAGGAGG CCAACGAGGG CGAGAACAACTGCGCCGCCC ACCCCATGTC

CCAGCACGGC ATCGAGGACC CCGAGAAGGA GGTGCTGGAGTGGAGGTTCG ACTCCAAGCT

IS GGCCTTCCAC CACGTGGCCA GGGAGCTGCA CCCCGAGTACTACAAGGACT GCTAAAGCCC

(SEQ ID N0:7).

The open reading frame of SEQ >D N0:7 encoding tPA-Nef (LLAA), disclosed herein as SEQ ID N0:8, is as follows:

Met Asp Ala Met Lys Arg Gly Leu Cys Leu Leu Leu Cys Gly Cys Val ZO Ala Val Phe Val Ser Pro Ser Glu Ile Lys Arg Ser Val Pro Ser Ser Gly Trp Ser Thr Val Arg Glu Arg Met Ala Glu Pro Ala Ala Arg Arg Asp Arg Val Arg Arg Thr Glu Pro Ala Gly Val Gly Ala Val Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ser Ser Asn Thr Ala Ile Thr Ala Thr Asn Ala Asp Cys Ala Trp Leu Gln Glu Asp Glu Glu Glu Ala ZS Val Gly Phe Pro Val Arg Pro Gln Val Arg Pro Met Thr Tyr Pro Leu Lys Gly Ala Val Asp Leu Ser His Phe Glu Lys Gly Gly Leu Leu Lys Glu Gly Leu Ile His Ser Gln Lys Arg Ile Leu Asp Leu Trp Gln Asp Val Tyr His Thr Gln Gly Tyr Phe Pro Gln Asn Tyr Thr Pro Asp Trp Gly Pro Gly Ile Arg Phe Pro Leu Thr Trp Cys Phe Lys Leu Phe Gly 30 Val Pro Val Glu Pro Glu Lys Val Glu Asn Glu Gly Glu Asn Glu Ala Asn Cys Ala Ala His Pro Met Ser Gln Ile Glu Asp Pro Glu His Gly Lys Glu Val Leu Glu Trp Arg Phe Asp Leu Ala Phe His His Ser Lys Val Ala Arg Glu Leu His Pro Glu Tyr Asp Cys (SEQ ID N0:8).
Tyr Lys The present invention also relates DNA molecule, regardless in part to any of codon usage, which expresses a wild type or modified Nef protein as described herein, including but not limited to modified Nef proteins which comprise a deletion or substitution of Gly 2, a deletion of substitution of Leu 174 and Leu 175 and/or inclusion of a leader sequence. Therefore, partial or fully codon optimized DNA
vaccine expression vector constructs are preferred since such constructs should result in increased host expression. However, it is within the scope of the present invention to utilize "non-codon optimized" versions of the constructs disclosed herein, especially modified versions of HIV Nef which are shown to promote a substantial cellular immune response subsequent to host administration.
The DNA backbone of the DNA vaccines of the present invention are preferably DNA plasmid expression vectors. DNA plasmid expression vectors are well known in the art and the present DNA vector vaccines may be comprised of any such expression backbone which contains at least a promoter for RNA polymerase transcription, and a transcriptional terminator 3' to the HIV nef coding sequence. In one preferred embodiment, the promoter is the Rous sarcoma virus (RSV) long terminal repeat (LTR) which is a strong transcriptional promoter. A more preferred promoter is the cytomegalovirus promoter with the intron A sequence (CMV-intA).
A preferred transcriptional terminator is the bovine growth hormone terminator. In addition, to assist in large scale preparation of an HIV nef DNA vector vaccine, an antibiotic resistance marker is also preferably included in the expression vector.
Ampicillin resistance genes, neomycin resistance genes or any other pharmaceutically acceptable antibiotic resistance marker may be used. In a preferred embodiment of this invention, the antibiotic resistance gene encodes a gene product for neomycin resistance. Further, to aid in the high level production of the pharmaceutical by fermentation in prokaryotic organisms, it is advantageous for the vector to contain an origin of replication and be of high copy number. Any of a number of commercially available prokaryotic cloning vectors provide these benefits. In a preferred embodiment of this invention, these functionalities are provided by the commercially available vectors known as pUC. It is desirable to remove non-essential DNA
sequences. Thus, the lacZ and lacI coding sequences of pUC are removed in one embodiment of the invention.
DNA expression vectors exemplified herein are also disclosed in PCT
International Application No. PCT/US94/02751, International Publication No. WO 94/21797, hereby incorporated by reference. A first DNA expression vector is the expression vector pnRSV, wherein the rous sarcoma virus (RSV) long terminal repeat (LTR) is used as the promoter. A second embodiment relates to plasmid V1, a mutated pBR322 vector into which the CMV promoter and the BGH transcriptional terminator is cloned. Another embodiment regarding DNA vector backbones relates to plasmid V1J. Plasmid V1J is derived from plasmid Vl and removes promoter and transcription termination elements in order to place them within a more defined context, create a more compact vector, and to improve plasmid purification yields.
Therefore, V1J also contains the CMVintA promoter and (BGH) transcription termination elements which control the expression of the HIV nef-based genes disclosed herein. The backbone of V1J is provided by pUCl8. It is known to produce high yields of plasmid, is well-characterized by sequence and function, and is of minimum size. The entire lac operon was removed and the remaining plasmid was purified from an agarose electrophoresis gel, blunt-ended with the T4 DNA
polymerise, treated with calf intestinal alkaline phosphatase, and ligated to the CMVintA/BGH element. In another DNA expression vector, the ampicillin resistance gene is removed from V1J and replaced with a neomycin resistance gene, to generate VlJneo. A DNA expression vector specifically exemplified herein is VlJns, which is the same as V 1J except that a unique Sfi 1 restriction site has been engineered into the single Kpn 1 site at position 2114 of V 1J-neo. The incidence of Sfi 1 sites in human genomic DNA is very low (approximately 1 site per 100,000 bases). Thus, this vector allows careful monitoring for expression vector integration into host DNA, simply by Sfi 1 digestion of extracted genomic DNA. Another DNA expression vector for use as the backbone to the HIV-1 nef-based DNA vaccines of the present invention is V
1R.
In this vector, as much non-essential DNA as possible is "trimmed" from the vector to produce a highly compact vector. This vector is a derivative of VlJns. This vector allows larger inserts to be used, with less concern that undesirable sequences are encoded and optimizes uptake by cells when the construct encoding specific influenza virus genes is introduced into surrounding tissue.
It will be evident upon review of the teaching within this specification that numerous vector/Nef antigen constructs may be generated. While the exemplified constructs (V lJns/nef, V lJns/tpanef, V lJns/tpanef(LLAA) and V
lJns/(G2A,LLAA) are preferred, any number of vector/Nef antigen combinations are within the scope of the present invention, especially wild type or modified Nef proteins which comprise a deletion or substitution of Gly 2, a deletion of substitution of Leu 174 and Leu 175 and/or inclusion of a leader sequence. Therefore, the present invention especially relates to DNA vaccines and a pharmaceutically active vaccine composition which contains this DNA vector vaccine, and the use as prophylactic and/or therapeutic vaccine for host immunization, preferably human host immunization, against an HIV
infection or to combat an existing HIV condition. These DNA vaccines are represented by codon optimized DNA molecules encoding HIV-1 Nef of biologically active Nef modifications or Nef-containing fusion proteins which are ligated within an appropriate DNA plasmid vector, with or without a nucleotide sequence encoding a functional leader peptide. DNA vaccines of the present invention include but in no way are limited to codon optimized DNA molecules encoding HIV-1 Nef of biologically active Nef modifications or Nef-containing fusion proteins ligated in DNA vectors Vl, V1J (SEQ >D N0:14), VlJneo (SEQ ID NO:15), VlJns (Figure 1A, SEQ ID N0:16), V1R (SEQ ID N0:26), or any of the aforementioned vectors wherein a nucleotide sequence encoding a leader peptide, preferably the human tPA
leader, is fused directly downstream of the CMV-intA promoter, including but not limited to V lJns-tpa, as shown in Figure 1B and SEQ ID N0:19. Especially preferred DNA vaccines of the present invention include as VlJns/nef, VlJns/tpanef, VlJns/tpanef(LLAA) and VlJns/(G2A,LLAA), as exemplified in Example Section 2.
The DNA vector vaccines of the present invention may be formulated in any pharmaceutically effective formulation for host administration. Any such formulation may be, for example, a saline solution such as phosphate buffered saline (PBS). It will be useful to utilize pharmaceutically acceptable formulations which also provide long-term stability of the DNA vector vaccines of the present invention.
During storage as a pharmaceutical entity, DNA plasmid vaccines undergo a physiochemical change in which the supercoiled plasmid converts to the open circular and linear form.
A variety of storage conditions (low pH, high temperature, low ionic strength) can accelerate this process. Therefore, the removal and/or chelation of trace metal ions (with succinic or malic acid, or with chelators containing multiple phosphate ligands) from the DNA plasmid solution, from the formulation buffers or from the vials and closures, stabilizes the DNA plasmid from this degradation pathway during storage.
In addition, inclusion of non-reducing free radical scavengers, such as ethanol or glycerol, are useful to prevent damage of the DNA plasmid from free radical production that may still occur, even in apparently demetalated solutions.
Furthermore, the buffer type, pH, salt concentration, light exposure, as well as the type of sterilization process used to prepare the vials, may be controlled in the formulation to optimize the stability of the DNA vaccine. Therefore, formulations that will provide the highest stability of the DNA vaccine will be one that includes a demetalated solution containing a buffer (phosphate or bicarbonate) with a pH
in the range of 7-8, a salt (NaCI, KCl or LiCI) in the range of 100-200 mM, a metal ion chelator (e.g., EDTA, diethylenetriaminepenta-acetic acid (DTPA), malate, inositol hexaphosphate, tripolyphosphate or polyphosphoric acid), a non-reducing free radical scavenger (e.g. ethanol, glycerol, methionine or dimethyl sulfoxide) and the highest appropriate DNA concentration in a sterile glass vial, packaged to protect the highly purified, nuclease free DNA from light. A particularly preferred formulation which will enhance long term stability of the DNA vector vaccines of the present invention would comprise a Tris-HCl buffer at a pH from about 8.0 to about 9.0; ethanol or glycerol at about 3°lo w/v; EDTA or DTPA in a concentration range up to about 5 mM; and NaCI at a concentration from about 50 mM to about 500 mM. The use of such stabilized DNA vector vaccines and various alternatives to this preferred formulation range is described in detail in PCT International Application No.
PCT/US97/06655, PCT International Publication No. WO 97/40839, which is hereby incorporated by reference.
The DNA vector vaccines of the present invention may, in addition to generating a strong CTL-based immune response, provide for a measurable humoral response subsequent immunization. This response may occur with or without the addition of adjuvant to the respective vaccine formulation. To this end, the DNA vector vaccines of the present invention may also be formulated with an adjuvant or adjuvants which may increase immunogenicity of the DNA
polynucleotide vaccines of the present invention. A number of these adjuvants are known in the art and are available for use in a DNA vaccine, including but not limited to particle bombardment using DNA-coated gold beads, co-administration of DNA vaccines with plasmid DNA expressing cytokines, chemokines, or costimulatory molecules, formulation of DNA with cationic lipids or with experimental adjuvants such as saponin, monophosphoryl lipid A or other compounds which increase immunogenicity of the DNA vaccine. One preferred adjuvant for use in the DNA vector vaccines of the present invention are one or more forms of an aluminum phosphate-based adjuvant. Aluminum phosphate is known in the art for use with live, killed or subunit vaccines, but is only recently disclosed as a useful adjuvant in DNA vaccine formulations. The artisan may alter the ratio of DNA to aluminum phosphate to provide for an optimal immune response. In addition, the aluminum phosphate-based adjuvant possesses a molar POa/Al ratio of approximately 0.9, and may again be altered by the skilled artisan to provide for an optimal immune response: An additional mineral-based adjuvant may be generated from one or more forms of a calcium phosphate. These mineral-based adjuvants are useful in increasing humoral responses to DNA
vaccination without imparting a negative effect on an appropriate cellular immune response. Complete guidance for use of these mineral-based compounds for use as DNA vaccines adjuvants are disclosed in PCT International Application No.
PCT/US98/02414, PCT International Publication No. WO 98/35562, which are hereby incorporated by reference in their entirety. Another preferred adjuvant is a non-ionic block copolymer which shows adjuvant activity with DNA vaccines.
The basic structure comprises blocks of polyoxyethylene (POE) and polyoxypropylene (POP) such as a POE-POP-POE block copolymer. Newman et al. (1998, Critical Reviews in Therapeutic Drug Carrier Systems 15(2): 89-142) review a class of non-ionic block copolymers which show adjuvant activity. The basic structure comprises blocks of polyoxyethylene (POE) and polyoxypropylene (POP) such as a POE-POP-POE block copolymer. Newman et al. id., disclose that certain POE-POP-POE block copolymers may be useful as adjuvants to an influenza protein-based vaccine, namely higher molecular weight POE-POP-POE
block copolymers containing a central POP block having a molecular weight of over about 9000 daltons to about 20,000 daltons and flanking POE blocks which comprise up to about 20% of the total molecular weight of the copolymer (see also U.S. Reissue Patent No. 36,665, U.S. Patent No. 5,567,859, U.S. Patent No.
5,691,387, U.S. Patent No. 5,696,298 and U.S. Patent No. 5,990,241, all issued to Emanuele, et al., regarding these POE-POP-POE block copolymers).
WO 96/04932 further discloses higher molecular weight POE/POP block copolymers which have surfactant characteristics and show biological efficacy as vaccine adjuvants. The above cited references within this paragraph are hereby incorporated by reference in their entirety. It is therefore within the purview of the skilled artisan to utilize available adjuvants which may increase the immune response of the polynucleotide vaccines of the present ivention in comparison to administration of a non-adjuvanted polynucleotide vaccine.

The DNA vector vaccines of the present invention are administered to the host by any means known in the art, such as enteral and parenteral routes. These routes of delivery include but are not limited to intramusclar injection, intraperitoneal injection, intravenous injection, inhalation or intranasal delivery, oral delivery, sublingual administration, subcutaneous administration, transdermal administration, transcutaneous administration, percutaneous administration or any form of particle bombardment, such as a biolostic device such as a "gene gun" or by any available needle-free injection device. The preferred methods of delivery of the HIV-1 Nef-based DNA vaccines disclosed herein are intramuscular injection and needle-free injection. An especially preferred method is intramuscular delivery.
The amount of expressible DNA to be introduced to a vaccine recipient will depend on the strength of the transcriptional and translational promoters used in the DNA construct, and on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of about 1 ~.g to greater than about 20 mg, and preferably in doses from about 1 mg to about 5 mg is administered directly into muscle tissue. As noted above, subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, inhalation and oral delivery are also contemplated. It is also contemplated that booster vaccinations are to be provided in a fashion which optimizes the overall immune response to the Nef-based DNA vector vaccines of the present invention.
The aforementioned polynucleotides, when directly introduced into a vertebrate in vivo, express the respective HIV-1 Nef protein within the animal and in turn induce a cytotoxic T lymphocyte (CTL) response within the host to the expressed Nef antigen. To this end, the present invention also relates to methods of using the HIV-1 Nef-based polynucleotide vaccines of the present invention to provide effective immunoprophylaxis, to prevent establishment of an HIV-1 infection following exposure to this virus, or as a post-HIV infection therapeutic vaccine to mitigate the acute HIV-1 infection so as to result in the establishment of a lower virus load with beneficial long term consequences. As noted above, the present invention contemplates a method of administration or use of the DNA nef-based vaccines of the present invention using an any of the known routes of introducing polynucleotides into living tissue to induce expression of proteins.
Therefore, the present invention provides for methods of using a DNA nef-based vaccine utilizing the various parameters disclosed herein as well as any additional parameters known in the art, which, upon introduction into mammalian tissue induces in vivo, intracellular expression of these DNA nef-based vaccines. This intracellular expression of the Nef-based immunogen induces a CTL and humoral response which provides a substantial level of protection against an existing infection or provides a substantial level of protection against a future infection in a presently uninfected host.
The following examples are provided to illustrate the present invention without, however, limiting the same hereto.

Vaccine Vectors Vl - Vaccine vector V 1 was constructed from pCMVIE-AKI-DHFR (Whang et al., 1987, J. Virol. 61: 1796). The AKI and DHFR genes were removed by cutting the vector with EcoRI and self-ligating. This vector does not contain intron A
in the CMV promoter, so it was added as a PCR fragment that had a deleted internal SacI
site [at 1855 as numbered in Chapman, et al., (1991, Nuc. Acids Res. 19:
3979)]. The template used for the PCR reactions was pCMVintA-Lux, made by ligating the HindIII and NheI fragment from pCMV6a120 (see Chapman et al., ibid.), which includes hCMV-IEl enhancer/promoter and intron A, into the HindIII and XbaI
sites of pBL3 to generate pCMVIntBL. The 1881 base pair luciferase gene fragment (HindIII-SmaI Klenow filled-in) from RSV-Lux (de Wet et al., 1987, Mol. Cell Biol.

7: 725) was ligated into the SaII site of pCMVIntBL, which was Klenow filled-in and phosphatase treated. The primers that spanned intron A are: 5' primer:
5'-CTATATAAGCAGAGCTCGTTTAG-3' (SEQ m NO:10); 3' primer:
5'-GTAGCAAAGATCTAAGGACGGTGACTGCAG-3' (SEQ >D NO:11). The primers used to remove the SacI site are: sense primer, 5'-GTATGTGTCTG
AAAATGAGC GTGGAGATTGGGCTCGCAC-3' (SEQ >D N0:12) and the antisense primer, 5'-GTGCGAGCCCAATCTCCACGCTCATTTTCAGAC
ACATAC-3' (SEQ >D N0:13). The PCR fragment was cut with Sac I and Bgl II and inserted into the vector which had been cut with the same enzymes.
V1J- Vaccine vector V1J was generated to remove the promoter and transcription termination elements from vector Vl in order to place them within a more defined context, create a more compact vector, and to improve plasmid purification yields. V1J is derived from vectors V1 and pUCl8, a commercially available plasmid. V1 was digested with SspI and EcoRI restriction enzymes producing two fragments of DNA. The smaller of these fragments, containing the CMVintA promoter and Bovine Growth Hormone (BGH) transcription termination S elements which control the expression of heterologous genes, was purified from an agarose electrophoresis gel. The ends of this DNA fragment were then "blunted"
using the T4 DNA polymerise enzyme in order to facilitate its ligation to another "blunt-ended" DNA fragment. pUClB was chosen to provide the "backbone" of the expression vector. It is known to produce high yields of plasmid, is well-characterized by sequence and function, and is of small size. The entire lac operon was removed from this vector by partial digestion with the HaeII restriction enzyme.
The remaining plasmid was purified from an agarose electrophoresis gel, blunt-ended with the T4 DNA polymerise treated with calf intestinal alkaline phosphatase, and ligated to the CMVintA/BGH element described above. Plasmids exhibiting either of 1S two possible orientations of the promoter elements within the pUC backbone were obtained. One of these plasmids gave much higher yields of DNA in E. coli and was designated V 1J. This vector's structure was verified by sequence analysis of the junction regions and was subsequently demonstrated to give comparable or higher expression of heterologous genes compared with V1. The nucleotide sequence of is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG

TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC
GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG
CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC
CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC

TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC
TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA
CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA
CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA

CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA

TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT

TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGC

S TTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTT

ATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCC

CTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCT

TTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC

AGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGC

ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC

CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA

CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC

TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC

IS GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC

CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC

GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT

CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC

CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA

ZO ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG

GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG

GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC

AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC

CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC

GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG

TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG

CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA

TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC

CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC

CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC

GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT

AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC

GTTCAGCCCGACCGCTGCGCCTTATCCGGT TTGAGTCCAACCCGGTAAGA
AACTATCGTC

CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA

GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA

TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA

S TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG

CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG

TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC

TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT

TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT

CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA

TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC

GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT

AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT

IS ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG

TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA

GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTA

AGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGG

CGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT

ZO TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG

CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT

ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA

ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC

ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA

ZS CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATT

ATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC (SEQ
ID

N0:14).

VlJneo vector involved - Construction of vaccine vector V lJneo expression removal of the ampr gene and insertion of the kanr gene (neomycin 30 phosphotransferase). The ampr gene from the pUC backbone of V1J was removed by digestion with SspI and Eam110SI restriction enzymes. The remaining plasmid was purified by agarose gel electrophoresis, blunt-ended with T4 DNA polymerase, and then treated with calf intestinal alkaline phosphatase. The commercially available kanr gene, derived from transposon 903 and contained within the pUC4K plasmid, was excised using the PstI restriction enzyme, purified by agarose gel electrophoresis, and blunt-ended with T4 DNA polymerise. This fragment was ligated with the V1J
backbone and plasmids with the kanr gene in either orientation were derived which were designated as VlJneo #'s 1 and 3. Each of these plasmids was confirmed by S restriction enzyme digestion analysis, DNA sequencing of the junction regions, and was shown to produce similar quantities of plasmid as V1J. Expression of heterologous gene products was also comparable to V1J for these VlJneo vectors.
VlJneo#3, referred to as VlJneo hereafter, was selected which contains the kanr gene in the same orientation as the ampr gene in V1J as the expression construct and provides resistance to neomycin, kanamycin and 6418. The nucleotide sequence of V lJneo is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
IS ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG
CTATTGGCCA TTGCATACGT TGTATCCATA TCATAATATG TACATTTATA TTGGCTCATG
TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC
GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG
CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC
ZO CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC
TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA
TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC
TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA
CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA

CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG
AGCTCGTTTA GTGAACCGTC AGATCGCCTG GAGACGCCAT CCACGCTGTT TTGACCTCCA
TAGAAGACAC CGGGACCGAT CCAGCCTCCG CGGCCGGGAA CGGTGCATTG GAACGCGGAT
TCCCCGTGCC AAGAGTGACG TAAGTACCGC CTATAGAGTC TATAGGCCCA CCCCCTTGGC

ATAGGTGATG GTATAGCTTA GCCTATAGGT GTGGGTTATT GACCATTATT GACCACTCCC
CTATTGGTGA CGATACTTTC CATTACTAAT CCATAACATG GCTCTTTGCC ACAACTCTCT
TTATTGGCTA TATGCCAATA CACTGTCCTT CAGAGACTGA CACGGACTCT GTATTTTTAC
AGGATGGGGT CTCATTTATT ATTTACAAAT TCACATATAC AACACCACCG TCCCCAGTGC

CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG

ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC

CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA

CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC

S TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC

GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC

CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC

GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT

CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC

ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG

GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG

GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC

AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC

IS CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC

TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG

GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG

TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG

CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA

ZO TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC

AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG

CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC

CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC

GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT

ZS AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC

GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA

CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA

GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA

TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA

CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG

TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC

TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT

TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT

CGTTCATCCATAGTTGCCTGACTCCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGA

AGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGA

GCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTT

TGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA

S AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT

TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT

TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGA

GAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG

ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGT

TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC

AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA

GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA

ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATC

IS GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGA

GGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG

CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAG

ATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCA

TCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATA

TTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCC

CATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT

TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

TAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT

2S CGTC (SEQ ID N0:15).
VIJns - The expression vector VIJns was generated by adding an SfiI site to VlJneo to facilitate integration studies. A commercially available 13 base pair SfiI
linker (New England BioLabs) was added at the KpnI site within the BGH
sequence of the vector. V lJneo was linearized with KpnI, gel purified, blunted by T4 DNA
30 polymerase, and ligated to the blunt SfiI linker. Clonal isolates were chosen by restriction mapping and verified by sequencing through the linker. The new vector was designated V lJns. Expression of heterologous genes in V lJns (with SfiI) was comparable to expression of the same genes in VlJneo (with KpnI).
The nucleotide sequence of VlJns is as follows:

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA

CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG

TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC

ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGG

S CTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATG

TCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC

GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG

CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC

IO TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA

TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC

TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA

CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA

CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA

IS CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA

TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT

TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGC

TCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA

ZO TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCC

TATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTC

TATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA

GGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCC

CGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGA

ZS CATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCC

AGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCAC

AGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCT

GAAAATGAGCGTGGAGATTGGGCTCGCACGGCTGACGCAGATGGAAGACTTAAGGCAGCG

GCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCC

CGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTC

TGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC

CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA

TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG

GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG

CTCTATGGCCGCTGCGGCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAG

AAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCA

GCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAA

S GTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGA

GTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAA

CATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTC

GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG

GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAA

IO GGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA

CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG

ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT

TACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG

CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC

IS CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT

AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA

TGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAAC

AGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC

TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT

ZO TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC

TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT

CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA

AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT

ATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGA

ZS AGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAG

GGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTG

CTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGC

AAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAG

TGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGC

GGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATT

CCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCA

AGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATT

TCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCA

ACCAAACCGT TATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA

AAAGGACAAT TACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCA

ACAATATTTT CACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGG

ATCGCAGTGG TGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGA

S AGAGGCATAA ATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCA

ACGCTACCTT TGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGA

TAGATTGTCG CACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCA

GCATCCATGT TGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTC

ATAACACCCC TTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATA

CCCCATTATT GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT

ATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC

GTCTAAGAAA CCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCC

TTTCGTC(SEQ ID N0:16).

1S The underlined nucleotides of SEQ ID N0:16 represent the Sfil site introduced into ite of the Kpn 1 s V lJneo.

VlJns-tPA - The V lJns-tPA in order vaccine vector was constructed to fuse an heterologous leader peptide sequence to the nef DNA constructs of the present invention. More specifically, the vaccine vector V lJns was modified to include the 20 human tissue-specific plasminogen activator (tPA) leader. As an exemplification, but by no means a limitation of generating a nef DNA construct comprising an amino-terminal leader sequence, plasmid V lJneo was modified to include the human tissue-specific plasminogen activator (tPA) leader. Two synthetic complementary oligomers were annealed and then legated into VlJneo which had been BgIII digested. The 2S sense and antisense oligomers were S' GATCACCATGGATGCAATGAAGAGAG
GGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAG
CGA-3' (SEQ ID N0:17); and, S'-GATCTCGCTGGGCGAAACGAAGACTGC
TCCACACAGCAGCAGCACACAGCAGAGCCCTCTCTTCATTGCATCCAT
GGT-3' (SEQ ID N0:18). The Kozak sequence is underlined in the sense oligomer.
30 These oligomers have overhanging bases compatible for legation to BgIII-cleaved sequences. After legation the upstream BgIII site is destroyed while the downstream BgIII is retained for subsequent legations. Both the junction sites as well as the entire tPA leader sequence were verified by DNA sequencing. Additionally, in order to conform with VlJns (=VlJneo with an SfiI site), an SfiI restriction site was placed at the KpnI within site the BGH
terminator region of V
lJneo-tPA
by blunting the KpnI

site with DNA polymerise by ligationth an T4 followed wi SfiI
linker (catalogue #1138, England New Biolabs), resulting in V
lJns-tPA.
This modification was verified by restriction digestion and agarose gel electrophoresis.

S The V lJns-tpa ce is as s:
vector follow nucleotide sequen TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA

CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG

TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC

ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGG

TCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC

GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG

CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC

IS TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA

TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC

TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA

CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA

CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA

ZO CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA

TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT

TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGC

TCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA

ZS TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCC

TATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTC

TATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA

GGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCC

CGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGA

AGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCAC

AGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCT

GAAAATGAGCGTGGAGATTGGGCTCGCACGGCTGACGCAGATGGAAGACTTAAGGCAGCG

GCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCC

GTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCG

CGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTC

TGCAGTCACCGTCCTTAGATCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTG

CTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCGAGATCTGCTGTGCCTTCTAGTTGCCA

S GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC

TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT

TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA

TGCTGGGGATGCGGTGGGCTCTATGGCCGCTGCGGCCAGGTGCTGAAGAATTGACCCGGT

TCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGC

IO CCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCT

TCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAAC

CAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGG

GAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGC

TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA

IS CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG

AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA

TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA

CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC

TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC

ZO GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT

GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG

TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG

GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA

CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG

ZS AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT

TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT

TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG

ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT

CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC

CTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATC

ATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTT

GGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGAT

CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTC

AGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAA
AAACTCATCG

AGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAA

AGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCC

TGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCG

S TCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAAT

GGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCA

TCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA

AATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGG

AACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGG

IO AATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATA

AAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCA

TCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCG

GGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCAT

TTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTT

IS TCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTT

ATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACA

ACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGC

GGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC

CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAAT

ZO AGGCGTATCACGAGGCCCTTTCGTC ID N0:9).
(SEQ

The u nderlinedleotides nuc of SEQ
ID N0:9 represent the Sfi 1 site introduced into the 1 site eo while icized Kpn of V the underlined/ital nucleotides IJn represent the human tPA
leader sequence.

V1 R - Vaccine onstructedobtain vector to a minimum-sized was c 2S vaccine vector without unneeded DNA sequences, which still retained the overall optimized heterologous gene expression characteristics and high plasmid yields that V1J and VlJns afford. It was determined that (1) regions within the pUC
backbone comprising the E. coli origin of replication could be removed without affecting plasmid yield from bacteria; (2) the 3'-region of the kanr gene following the 30 kanamycin open reading frame could be removed if a bacterial terminator was inserted in its place; and, (3) 300 by from the 3'- half of the BGH terminator could be removed without affecting its regulatory function (following the original KpnI
restriction enzyme site within the BGH element). V1R was constructed by using PCR
to synthesize three segments of DNA from VlJns representing the CMVintA

promoter/BGH terminator, origin of replication, and kanamycin resistance elements, respectively. Restriction enzymes unique for each segment were added to each segment end using the PCR oligomers: SspI and XhoI for CMVintABGH; EcoRV
and BamHI for the kan r gene; and, BcII and SaII for the on r. These enzyme sites were chosen because they allow directional ligation of each of the PCR-derived DNA
segments with subsequent loss of each site: EcoRV and SspI leave blunt-ended DNAs which are compatible for ligation while BamHI and BcII leave complementary overhangs as do SaII and XhoI. After obtaining these segments by PCR each segment was digested with the appropriate restriction enzymes indicated above and then ligated together in a single reaction mixture containing all three DNA
segments. The 5'-end of the on r was designed to include the T2 rho independent terminator sequence that is normally found in this region so that it could provide termination information for the kanamycin resistance gene. The ligated product was confirmed by restriction enzyme digestion (>8 enzymes) as well as by DNA sequencing of the ligation junctions. DNA plasmid yields and heterologous expression using viral genes within V 1R appear similar to V lJns. The net reduction in vector size achieved was 1346 by (V lJns = 4.86 kb; V 1R = 3.52 kb). PCR oligomer sequences used to synthesize V1R (restriction enzyme sites are underlined and identified in brackets following sequence) are as follows: (1) 5'-GGTACAAATATTGGCTATTGGC
CATTGCATACG-3' (SEQ >D N0:20) [SspI]; (2) 5'-CCACATCTCGAGGAA
CCGGGTCAATTCTTCAGCACC-3' (SEQ >D N0:21) [XhoI] (for CMVintA/BGH
segment); (3) 5'-GGTACAGATATCGGAAAGCCACGTTGTG TCTCAAAATC-3' (SEQ >D N0:22) [EcoRV]; (4) 5'-CACATGGATCCGTAATGCTCTGCCAGTGT
TACAACC-3' (SEQ ID N0:23) [BamHI], (for kanamycin resistance gene segment) (5) 5'-GGTACATG ATCACGTAGAAAAGATCAAAGGATCTTCTTG-3' (SEQ >D
N0:24) [BcII]; (6) 5'-CCACATGTCGACCCGTAAAAAGGCCGCGTTGCTGG-3' (SEQ >D N0:25): [SaII], (for E. coli origin of replication).
The nucleotide sequence of vector V1R is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA

TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG
CTATTGGCCA TTGCATACGT TGTATCCATA TCATAATATG TACATTTATA TTGGCTCATG
TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC

GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG

CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC

TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA

S TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC

TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA

CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA

CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA

CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

IO AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA

TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT

TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGC

TTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTT

ATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCC

IS CTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCT

TTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC

AGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGC

CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG

ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC

ZO CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA

CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC

TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC

GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC

CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC

ZS GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT

CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC

CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA

ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG

GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG

AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC

CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC

TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG

GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG

TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG

CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA

TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC

AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG

S CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC

CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC

GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT

AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC

GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA

GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA

TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA

TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG

CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG

IS TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC

TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT

TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT

CGTTCATCCATAGTTGCCTGACTCCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGA

AGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGA

TGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA

AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT

TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT

TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGA

ZS GAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG

ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGT

GAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCT

TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC

AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA

ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATC

GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGA

GGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG

CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAG

ATTGTCGCAC CTGATTGCCC GACATTATCG CGAGCCCATT TATACCCATA TAAATCAGCA
TCCATGTTGG AATTTAATCG CGGCCTCGAG CAAGACGTTT CCCGTTGAAT ATGGCTCATA
ACACCCCTTG TATTACTGTT TATGTAAGCA GACAGTTTTA TTGTTCATGA TGATATATTT
TTATCTTGTG CAATGTAACA TCAGAGATTT TGAGACACAA CGTGGCTTTC CCCCCCCCCC
S CATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT
TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC
TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT
CGTC (SEQ ID N0:26).

Codon Optimized HIV-1 Nef and HIV-1 Nef Derivatives as DNA Vector Vaccines HIV-1 Nef Vaccine Vectors - Codon optimized nef gene coding for wt Nef protein of HIV-1 jrfl isolate was assembled from complementary, overlapping synthetic oligonucleotides by polymerise chain reaction (PCR). The PCR primers 1S used were designed in such that a BgIII site was included in the extension of S' primer and an SrfI site and a BgIII site in the extension of 3' primer. The PCR
product was digested with BgIII and cloned into BgIII site of a human cytomeglovirus early promoter-based expression vector, VlJns (Figure 1A). The proper orientation of nef fragment in the context of the expression cassette was determined by asymmetric restriction mapping. The resultant plasmid is VlJns/nef. The S' and 3' nucleotide sequence junctions of codon optimized VlJns/nef are shown in Figure 3A.
The mutant nef (G2A,LLAA) was also made from synthetic oligonucleotides.
To assist in cloning, a PstI site and an SrfI site were included in the extensions of S' and 3' PCR primers, respectively. The PCR product was digested with PstI and SrfI, 2S and cloned into the PstI and Srfl sites of VlJns/nef, replacing the original nef with nef(G2A,LLAA) fragment. This resulted in VlJns/nef(G2A,LLAA). The S' and 3' nucleotide sequence junctions of codon optimized VlJns/nef (G2A,LLAA) are shown in Figure 3B.
To construct the expression vector containing human tissue plasminogen activator leader peptide and the nef fusion gene, i.e., VlJns/tPAnef, a truncated nef gene fragment, lacking the coding sequence for the five amino terminal residues, was first amplified by PCR using VlJns/nef as template. Both S' and 3' PCR primers used in this reaction contained a BgIII extension. The PCR amplified fragment was then digested with BgIII and cloned into BgIII site of the expression vector, VlJns/tpa (Figure 1B). The ligation of the 3' end of tpa leader peptide coding sequence to the 5' end of the nef PCR product restored the BgIII site and yielded an in-frame fusion of the two genes. The 5' and 3' nucleotide sequence junctions of codon optimized VlJns/tPAnef are shown in Figure 3C.
Construction of V lJns/tpanef(LLAA) was carried out by replacing the Bsu36-SacII fragment of VlJns/tpanef, which contains the 3' half of the nef gene and part of the vector backbone, with the Bsu36-SacII fragment from V lJns/nef(G2A,LLAA).
The 5' and 3' nucleotide sequence junctions of codon optimized V lJns/tpanef (LLAA) are shown in Figure 3C.
All the nef constructs were verified by sequencing. The amino acid junctions of these constructs is shown schematically in Figure 4.
Transfection and protein expression - 293 cells (adenovirus transformed human embryonic kidney cell line 293) grown at approximately 30% confluence in minimum essential medium (MEM; GIBCO, Grand Island, MD) supplemented with 10% fetal bovine serum (FBS; GIBCO) in a 100 mm culture dish, were transfected with 4 ug gag expression vector, VlJns/gag, or a mixture of 4 ug gag expression vector and 4 ug nef expression vector by Lipofectin following manufacture's protocol (GIBCO). Twelve hours post-transfection, cells were washed once with 10 ml of serum-free medium, Opti-MEM I (GIBCO) and replenished with 5 ml of Opti-MEM.
Following an additional 60 hr incubation, culture supernatants and cells were collected separately and used for Western blot analysis.
Western blot analysis - Fifty microliter of samples were separated on a 10%
SDS-polyacrylamide gel (SDS-PAGE) under reducing conditions. The proteins were blotted onto a piece of PVDF membrane, and reacted to a mixture of gag mAb (#18;
Intracel, Cambridge, MA) and Nef mAbs (aa64-68, aa195-201; Advanced Biotechnologies, Columbia, MD), both at 1:2000 dilution, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA). The protein bands were visualized by ECL Western blotting detection reagents, according to the manufacture's protocol (Amersham, Arlington Heights, IL).
Enzyme-linked immunosorbent assay (ELISA) - 96-well Immulon II, round-bottom plates were coated with 50 u1 of Nef protein at the concentration of 2ug/ml in bicarbonate buffer, pH 9.8., per well at 4°C overnight. Plates were washed three times with PBS containing 0.05°lo Tween-20 (PBST), and blocked with 5°Io skim milk in PBST (milk-PBST) at 24°C for 2 hr, and then incubated with serial dilutions of testing samples in milk-PBST at 24°C for 2 hr. Plates were washed with PBST three times, and added with 50 u1 of HRP-conjugated goat anti-mouse IgG (Zymed) per well and incubated at 24°C for 1 hr. This was followed by three washes, and the addition of 100 u1 of 1 mg/ml ABTS [(2,2'-amino-di-(3-ethylbenzthiozoline sulfonate)] (KPL, Gaithersburg, MD) per well. After 1 hr at 24°C, plates were read at a wavelength of 405nm using an ELISA plate reader.
Enzyme-linked spot assay (Elispot) - Nitrocellulose membrane-backed 96 well plates (MSHA plates; Millipore, Bedford, MA) were coated with 50 u1 of rat anti-mouse IFN-gamma mAb, capture antibody, (R4-6A2; PharMingen, San Diego, CA) at a concentration of Sug/ml in PBS per well at 4°C overnight. Plates were washed three times with PBST and blocked with 10% FBS in RPMI-1640 (FBS-RPMI) at 37°C in a C02 incubator for 2 to 4 hrs. Splenocytes were suspended in RPMI-1640 with 10%
FBS at 4 x 10G cells per ml. 100 u1 cells were added to each well and plates were incubated at 37°C for 20 hrs. Each sample was tested in triplicate wells. After incubation, plates were rinsed briefly with distilled water and washed three times with PBST. Fifty u1 of biotinylated rat anti-mouse IFN-y mAb, detecting antibody (XMG1.2; PharMingen), diluted in 1% BSA in PBST at a concentration of 2 ug/ml was then added to each well. Plates were incubated at 24°C for 2 hr, followed by washes with PBST. Fifty u1 of streptavidin-conjugated alkaline phosphatase (KPL) at a dilution of 1:1000 in FBS-RPMI was added to each well. The plates were incubated at 24C for an additional one hr. Following extensive wash with BPST, 100u1 BCIT/NBT substrate (KPL) was added for 15 min, and color reaction was stopped by washing the plate with tap water. Plates were air-dried and spots were countered using a dissection microscope.
Cytotoxic T cell (CTL) assay - Splenocytes from immunized mouse were co-cultured with syngenic peptide-pulsed, irradiated naive splenocytes for 7 days. EL-4 cells were incubated at 37°C for 1 hr with or without 20ug/ml of a designated peptide in the presence of sodium SICr-chromate and used as target cells. For the assay, 104 target cells were added to a 96-well plate along with different numbers of splenocytes cells. Plates were incubated at 37°C for 4 hr. After incubation, supernatants were collected and counted in a Wallac gamma-counter. Specific lysis was calculated as ([experimental release - spontaneous release]/maximum release- spontaneous release]) x 100%. Spontaneous release was determined by incubating target cells in medium alone, and maximum release was determined by incubating target cells in 2.5% TritonX-100. The assay was performed with triplicate samples.
Animal experiments - Female mice (Charles River Laboratories, Wilmington, MA), 6 to 10 weeks old, were injected in quadriceps with 100 u1 of DNA in PBS.
Two weeks after immunization, spleens from individual mice were collected and used for CTL and Elispot assays.
Results (DNA Vector Vaccine Construction) - The exemplified Nef protein sequence is based on HIV-1 Glade B jrfl isolate. A codon-optimized nef gene was chosen for vaccine construction and for use as the parental gene for other exemplified constructs. Figure 2A-B show the comparison of coding sequence of wt nef(jrfl) and the codon optimized nef(jrfl). Two forms of myristylation site mutations were constructed; one contains a Gly2Ala change and the other a human tissue plasminogen activator (tpa) leader sequence was fused to sixth residue, Ser, of Nef (tpanef). The dileucine motif mutation was made by introducing both Leu174A1a and Leu175A1a changes. Figure 4 shows the schematic depiction of the Nef and Nef mutants. For in vitro expression and in vivo immunogenicity studies, the nef genes were cloned into expression vector, VlJns. The resultant plasmids containing wt nef, tpanef, tpanef with dileucine motif mutation, and nef mutant with the Gly2Ala myristylation site and dileucine motif mutations were named as V lJns/nef, VlJns/tpanef, VlJns/tpanef(LLAA) and VlJns/(G2A,LLAA), respectively.
Results - Expression and Western blotting analysis - To evaluate the expression of the codon optimized nef constructs, adenovirus-transformed human kidney 293 cells were cotransfected with individual nef plasmids and a gag expression vector, VlJns/gag. 72 hours post transfection, cells and medium were collected separately and analyzed by Western blotting, using both Nef- and Gag-specific mAbs.
The results are shown in Figure 5. Cells transfected with V lJns/gag only revealed a single distinct band of approximately 55 Kd, whereas the cells cotransfected with gag and nef plasmids revealed, in addition to the 55 Kd band, a major 30 Kd band and several minor bands. This pattern is consistent with that the 55 Kd species represents Gag polypeptide and the 30 Kd and other minor species are the Nef-related products.
Therefore, all the nef constructs were expressed in the transfected cells.
When measured against the relatively constant Gag signal as a reference, four nef genes seem to be expressed at different levels, with the following descending order, tpanef, nef, tpanef(LLAA) and nef(G2A, LLAA). With the exception of nef(G2A,LLAA), products of nef, tpanef, tpanef(LLAA) could be detected in both cellular and medium fractions.
Mapping of Nef specific CD8 and CD4 epitopes in mice - There was no information available with respect to the properties of Nef(jrfl) in eliciting cell-mediated immune responses in mice. Therefore, to characterize immunogenicity of Nef and Nef mutants exemplified herein, CD8 and CD4 epitopes were mapped. An overlapping set of overlapping nef peptides that encompass the entire 216 as Nef polypeptide were generated. A total 21 peptides were made, which include twenty 20mers and one l6mer. Three strains of mice, Balb/c, C3H and C57BL/6, were immunized with plasmid VlJns/Nef; splenocytes from immunized and naive mice were isolated and assessed for Nef specific INF-gamma secreting cells (SFC) by the Elispot assay. Figure 6 shows where Elispot assays were performed against separate pools of the Nef peptides. All three strains of immunized mice responded to the Nef plasmid immunization; each developed positive Nef peptide-specific INF-'y SFCs.
Based on this, further studies were carried out with fractionated CD8 and CD4 cells against individual peptides. The results are shown in Figure 7A-C. In Balb/c mice (Figure 7A), four Nef peptides, namely, aal l-30, aa61-80, aa191-210 and aa200-216, were found to be able to induce significant numbers of CD4 SFCs. In C57BL/6 mice (Figure 7B), only one peptide, ie., aa81-100, elicited significant numbers of SFCs. Compared to Balb/c and C57BL/6 mice, C3H mice (Figure 7C) showed no dominant CD4 SFC responses with particular peptides; instead, there were modest number of SFCs in response to an array of peptides, including aa21-40, aa31-50, aa121-140 aa131-150, aa181-200 and aa191-210. With respect to CD8 cells, significant SFC responses were detected with a single peptide, ie., aa51-70, in C57BL/6 mice only.
The results from Elispot assay suggested that Nef peptide aa51-70 contained an H-2b restricted CD8 cell epitope. In order to ascertain whether this CD8 epitope also represents the cytotoxic T cell (CTL) epitope, a conventional CTL assay was carried out. The peptide aa51-70 (Figure 8A) induced low level of specific killings only. Peptides longer than 9 amino acids of a typical CTL epitope often have lower binding affinity to MHC class I molecule. It was contemplated that the low specific killings observed with peptide aa51-70 could be potentially resulted from the low binding affinity of this 20 amino acid peptide. Therefore, two shortened peptides, namely, aa60-68 and aa58-70, were synthesized and tested in CTL assays. While the peptide aa60-68 failed to elicit any specific killings (Figure 8B), the peptide aa58-70 exhibited a drastic increase of specific killing as compared to its longer counterpart, peptide aa61-80 (Figure 8C). For example, the percentage of specific killings induced by peptide aa58-70 at an effector/target ratio of 5 to 1 was comparable to that induced by peptide aa51-80 at an effector/target ratio of 45. Thus, between peptide aa58-70 and peptide aa51-70, the former was almost ten-fold more effective in terms of inducing Nef-specific killing. The results from CTL assay therefore confirmed that the CD8 epitope detected by the Elispot assay was indeed a CTL epitope. To further map the minimum amino acid sequence for the Nef CTL epitope, additional 5 peptides were synthesized and analyzed by Elispot assay, which mapped the CTL
epitope to Nef aa58-66, as shown in Table 1.

Nef peptides** INF-y SFC*/106 splenocytes Nef58-70 TAATNADCAWLEA 85 Nef59-69 AATNADCAWLE 1 Nef58-68 TAATNADCAWL 69 Nef58-67 TAATNADCAW 66 Nef58-66 TAATNADCA 92 Medium 1 * Average of duplicate samples.
** Amino acid sequence of all peptides contained within SEQ >D N0:2.

Results (Evaluation of Immunogenicity of nef Mutants in Mice) - Having identified H-2b restricted CTL and CD4 cell epitopes, the immunogenicity of the different codon optimized nef constructs in C57BL/6 mice was examined. This was performed in two separate experiments with identical immunization regimens.
The first experiment involved nef, tpanef(LLAA) and nef(G2A,LLAA) and the second experiment involved nef, tpanef, tpanef(LLAA) and nef(G2A,LLAA). Mice were immunized with plasmids containing these respective codon optimized nef genes.
Two weeks post immunization, splenocytes from individual mice were isolated and analyzed by Elispot assay for Nef-specific CD8 and CD4 IFN-gamma SFCs using Nef peptide aa58-66 and aa81-100, respectively. The results are shown in Figure 9A-B.
In the experiment 1 (Figure 9A), among the three groups tested, the mice receiving the codon optimized tpanef(LLAA) construct developed the highest CD8 and CD4 cell responses; comparing between tpanef(LLAA) and the nef, the former elicited about 40-fold higher CD8 SFCs and 10-fold higher CD4 SFCs. In contrast to tpanef(LLAA), nef(G2A,LLAA) mutant was poorly immunogenic; mice receiving this mutant had barely detectable CD8 and CD4 SFCS, under conditions tested.
Similar response profiles between the three mutants were also observed in the experiment 2 (Figure 9B), except that the overall CD8 response of mice receiving tpanef(LLAA) was approximately 10-folder higher in experiment 2 than that observed in experiment 1. The tPAnef mutant showed comparable responses as that of tpanef(LLAA). The results therefore showed that both codon optimized tpanef and tpanef(LLAA) had significantly enhanced immunogenicity.
Results (Evaluation of Immunogenicity of nef Mutants in Rhesus Monkeys) -Monkeys were immunized with 5 mg of indicated codon optimized plasmids at week 0, 4, and 8. Four weeks after each immunization , peripheral blood mononuclear cells were collected and tested for Nef-specific INF-gamma secreting cells as described for the mice studies in this Example section. The results are shown in Table 2. As with the mouse study, tpanef(LLAA) shows significantly enhanced immunogenicity when compared to tPAnef.

Nef specific INF-gamma secreting cells/million PBMC

A
al Vaccine Week Week Week Week No.

Medium Medium Medium Medium nef nef nef nef VlJns- 1 7439 30208 6 148 89 559 TpaNef 2 1 3 2845 1344 13 146 (LLAA) V lJns-nef1 0 1 2433 1643 6 34 Control1 1 3 1633 1616 18 13 Monkeys were immunized with 5 mg of indicated plasmids at week 0, 4 and 8.
Four weeks after each immunization, peripheral blood mononuclear cells were collected and tested for the Nef-specific IFN-gamma secreting cells.
A codon-optimized nef gene coding for HIV-1 jrfl isolate Nef polypeptide was synthesized. The resultant synthetic nef gene was well expressed in the in vitro transfected cells. Using this synthetic gene as parental molecule, nef mutants involving myristylation site and dileucine motif mutations were constructed.
Two forms of myristylation site mutation were made, one involving a single Gly2Ala change and the other by fusing human plasminogen activator(tpa) leader peptide with the N-terminus of Nef polypeptide. The dileucine motif mutation was generated by Leu174A1a and Leu175A1a changes. The resultant nef constructs were named as nef, tpanef, tpanef(LLAA) and nef(G2A,LLAA). The addition of tpa leader peptide sequence resulted in significantly increased expression of the nef gene in vitro; in contrast, either Gly2Ala mutation or dileucine mutation reduced the nef gene expression. In an effort to characterize immunogenicity of nef and nef mutants, experiments were carried out to map nef CTL and Th epitopes in mice. A single CTL
epitope and a dominant Th epitope, both restricted by H-2b, were identified.
Consequently, C57BL/6 mice were immunized with different nef constructs by DNA
immunization means, and splenocytes from immunized mice were determined for Nef-specific CTL and Th responses using Elisopt assay and the defined T cell epitopes. The results showed that tpanef and tpanef(LLAA) were significantly more immunogenic than nef in terms of eliciting both CTL and Th responses.
Therefore, these aforementioned polynucleotides, when directly introduced into a vertebrate in vivo, including mammals such as primates and humans, should express the respective HIV-1 Nef protein within the animal and in turn induce at least a cytotoxic T lymphocyte (CTL) response within the host to the expressed Nef antigen.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

SEQUENCE LISTING
<110> APPLICANT: Merck & Co., Inc.
<120> TITLE: POLYNUCLEOTIDE VACCINES EXPRESSING CODON

<130> DOCKET/FILE REFERENCE: 20602Y
<160> NUMBER OF SEQUENCES: 30 <170> SOFTWARE: FastSEQ for Windows Version 4.0 <210> SEQ ID N0:1 <211> LENGTH: 671 <212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1 <220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (12)...(662) <400> SEQ ID N0:1 gatctgccac c atg ggc ggc aag tgg tcc aag agg tcc gtg ccc ggc tgg 50 Met Gly Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc gac agg 98 Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg tcc agg 146 Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc gcc acc 194 Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag gtg ggc 242 Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac aag ggc 290 Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg gag ggc 338 Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly ctg atc cac tcc cag aag agg cag gac atc ctg gac ctg tgg gtg tac 386 Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr cac acc cag ggc tac ttc ccc gac tgg cag aac tac acc ccc ggc ccc 434 His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro ggc atc agg ttc ccc ctg acc ttc ggc tgg tgc ttc aag ctg gtg ccc 482 Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro gtg gag ccc gag aag gtg gag gag gcc aac gag ggc gag aac aac tgc 530 Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys ctg ctg cac ccc atg tcc cag cac ggc atc gag gac ccc gag aag gag 578 Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu gtg ctg gag tgg agg ttc gac tcc aag ctg gcc ttc cac cac gtg gcc 626 Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala agg gag ctg cac ccc gag tac tac aag gac tgc taa agcccgggc 671 Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys <210> SEQ ID N0:2 <211> LENGTH: 216 <212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1 <400> SEQ ID N0:2 Met Gly Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys <210> SEQ ID N0:3 <211> LENGTH: 719 <212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1 <220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (2)...(715) <400> SEQ ID N0:3 c atg gat gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt gga 49 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly gca gtc ttc gtt tcg ccc agc gag atc tcc tcc aag agg tcc gtg ccc 97 Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro ggc tgg tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc 145 Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala gac agg gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg 193 Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val tcc agg gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc 241 Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala gcc acc aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag 289 Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu gtg ggc ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac 337 Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr aag ggc gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg 385 Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu gag ggc ctg atc cac tcc cag aag agg cag gac atc ctg gac ctg tgg 433 Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp gtg tac cac acc cag ggc tac ttc ccc gac tgg cag aac tac acc ccc 481 Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro ggc ccc ggc atc agg ttc ccc ctg acc ttc ggc tgg tgc ttc aag ctg 529 Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu gtg ccc gtg gag ccc gag aag gtg gag gag gcc aac gag ggc gag aac 577 Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn aac tgc ctg ctg cac ccc atg tcc cag cac ggc atc gag gac ccc gag 625 Asn Cys Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu aag gag gtg ctg gag tgg agg ttc gac tcc aag ctg gcc ttc cac cac 673 Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His gtg gcc agg gag ctg cac ccc gag tac tac aag gac tgc taa 715 Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys agcc 719 <210> SEQ ID N0:4 <211> LENGTH: 237 <212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1 <400> SEQ ID N0:4 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys <210> SEQ ID N0:5 <211> LENGTH: 671 <212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1 <220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (12)...(662) <400> SEQ ID N0:5 gatctgccac c atg gcc ggc aag tgg tcc aag agg tcc gtg ccc ggc tgg 50 Met Ala Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc gac agg 98 Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg tcc agg 146 Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc gcc acc 194 Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag gtg ggc 242 Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac aag ggc 290 Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg gag ggc 338 Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly ctg atc cac tcc cag aag agg cag gac atc ctg gac ctg tgg gtg tac 386 Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr cac acc cag ggc tac ttc ccc gac tgg cag aac tac acc ccc ggc ccc 434 His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro ggc atc agg ttc ccc ctg acc ttc ggc tgg tgc ttc aag ctg gtg ccc 482 Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro gtg gag ccc gag aag gtg gag gag gcc aac gag ggc gag aac aac tgc 530 Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys gcc gcc cac ccc atg tcc cag cac ggc atc gag gac ccc gag aag gag 578 Ala Ala His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu gtg ctg gag tgg agg ttc gac tcc aag ctg gcc ttc cac cac gtg gcc 626 Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala agg gag ctg cac ccc gag tac tac aag gac tgc taa agcccgggc 671 Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys <210> SEQ ID N0:6 <211> LENGTH: 217 <212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1 <400> SEQ ID N0:6 Met Ala Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Ala Ala His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys Ser <210> SEQ ID N0:7 <211> LENGTH: 720 <212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1 <220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (2)...(715) <400> SEQ ID N0:7 c atg gat gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt gga 49 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly gca gtc ttc gtt tcg ccc agc gag atc tcc tcc aag agg tcc gtg ccc 97 Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro ggc tgg tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc 145 Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala gac agg gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg 193 Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val tcc agg gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc 241 Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala gcc acc aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag 289 Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu gtg ggc ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac 337 Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr aag ggc gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg 385 Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu gagggcctgatc cactcccag aagaggcag gacatcctg gacctgtgg 433 GluGlyLeuIle HisSerGln LysArgGln AspIleLeu AspLeuTrp gtgtaccacacc cagggctac ttccccgac tggcagaac tacaccccc 481 ValTyrHisThr GlnGlyTyr PheProAsp TrpGlnAsn TyrThrPro ggccccggcatc aggttcccc ctgaccttc ggctggtgc ttcaagctg 529 GlyProGlyIle ArgPhePro LeuThrPhe GlyTrpCys PheLysLeu gtgcccgtggag cccgagaag gtggaggag gccaacgag ggcgagaac 577 ValProValGlu ProGluLys ValGluGlu AlaAsnGlu GlyGluAsn aactgcgccgcc caccccatg tcccagcac ggcatcgag gaccccgag 625 AsnCysAlaAla HisProMet SerGlnHis GlyIleGlu AspProGlu aaggaggtgctg gagtggagg ttcgactcc aagctggcc ttccaccac 673 LysGluValLeu GluTrpArg PheAspSer LysLeuAla PheHisHis gtggccagggag ctgcacccc gagtactac aaggactgc taa 715 ValAlaArgGlu LeuHisPro GluTyrTyr LysAspCys agccc 720 <210> SEQ ID N0:8 <211> LENGTH: 237 <212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1 <400> SEQ ID N0:8 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Ala Ala His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys <210> SEQ ID N0:9 <211> LENGTH: 4945 <212> TYPE: DNA
<213> ORGANISM: E. coli <400> SEQ ID N0:9 tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60 cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120 ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180 accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240 ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300 tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360 ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420 cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540 tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600 tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660 ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720 catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780 cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840 ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900 agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960 tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020 tccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggc 1080 tcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgcta 1140 taggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccc 1200 tattggtgacgatactttccattactaatccataacatggctctttgccacaactatctc 1260 tattggctatatgccaatactctgtccttcagagactgacacggactctgtatttttaca 1320 ggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcc 1380 cgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccgga 1440 catgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctcc 1500 agcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcac 1560 agcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtct 1620 gaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcg 1680 gcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactccc 1740 gttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcg 1800 cgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttc 1860 tgcagtcaccgtccttagatcaccatggatgcaatgaagagagggctctgctgtgtgctg 1920 ctgctgtgtggagcagtcttcgtttcgcccagcgagatctgctgtgccttctagttgcca 1980 gccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccac 2040 tgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat 2100 tctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggca 2160 tgctggggatgcggtgggctctatggccgctgcggccaggtgctgaagaattgacccggt 2220 tcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgc 2280 ccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgcct 2340 tcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaac 2400 caaacctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagg 2460 gagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaatttcttccgc 2520 ttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca 2580 ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtg 2640 agcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca 2700 taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa 2760 cccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcc 2820 tgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggc 2880 gctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagct 2940 gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg 3000 tcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag 3060 gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta 3120 cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgg 3180 aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt 3240 tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt 3300 ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag 3360 attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat 3420 ctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacc 3480 tatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcg 3540 ctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatc 3600 atccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagtt 3660 ggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgat 3720 ctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtc 3780 agcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcg 3840 agcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaa 3900 agccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcc 3960 tggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcg 4020 tcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaat 4080 ggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtca 4140 tcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacga 4200 aatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcagg 4260 aacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctgg 4320 aatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggata 4380 aaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctca 4440 tctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcg 4500 ggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccat 4560 ttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtt 4620 tcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagtttt 4680 attgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacaca 4740 acgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagc 4800 ggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccc 4860 cgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaat 4920 aggcgtatcacgaggccctttcgtc 4945 <210> SEQ ID N0:10 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:10 ctatataagc agagctcgtt tag 23 <210> SEQ ID N0:11 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:11 gtagcaaaga tctaaggacg gtgactgcag 30 <210> SEQ ID N0:12 <211> LENGTH: 39 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:12 gtatgtgtct gaaaatgagc gtggagattg ggctcgcac 39 <210> SEQ ID N0:13 <211> LENGTH: 39 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:13 gtgcgagccc aatctccacg ctcattttca gacacatac 39 <210> SEQ ID N0:14 <211> LENGTH: 4432 <212> TYPE: DNA
<213> ORGANISM: E. coli <400> SEQ ID N0:14 tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60 cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120 ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180 accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240 ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300 tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360 ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420 cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540 tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600 tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660 ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720 catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780 cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840 ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900 agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960 tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020 tccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggc 1080 ttcttatgcatgctatactgtttttggcttggggtctatacacccccgcttcctcatgtt 1140 ataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactccc 1200 ctattggtgacgatactttccattactaatccataacatggctctttgccacaactctct 1260 ttattggctatatgccaatacactgtccttcagagactgacacggactctgtatttttac 1320 aggatggggtctcatttattatttacaaattcacatatacaacaccaccgtccccagtgc 1380 ccgcagtttttattaaacataacgtgggatctccacgcgaatctcgggtacgtgttccgg 1440 acatgggctcttctccggtagcggcggagcttctacatccgagccctgctcccatgcctc 1500 cagcgactcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggca 1560 cagcacgatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtc 1620 tgaaaatgagctcggggagcgggcttgcaccgctgacgcatttggaagacttaaggcagc 1680 ggcagaagaagatgcaggcagctgagttgttgtgttctgataagagtcagaggtaactcc 1740 cgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgc 1800 gcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtctttt 1860 ctgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgcc 1920 cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa 1980 atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgg 2040 ggcagcacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg 2100 gctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagc 2160 aggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccc 2220 cactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtac 2280 ttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgg 2340 gaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatg 2400 tgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctg 2460 cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta 2520 tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc 2580 aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag 2640 catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac 2700 caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc 2760 ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgt 2820 aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc 2880 gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga 2940 cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 3000 ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagta 3060 tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga 3120 tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg 3180 cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag 3240 tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc 3300 tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 3360 tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt 3420 cgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctta 3480 ccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta 3540 tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatcc 3600 gcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaat 3660 agtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggt 3720 atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttg 3780 tgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca 3840 gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgta 3900 agatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcgg 3960 cgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaact 4020 ttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg 4080 ctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt 4140 actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggga 4200 ataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagc 4260 atttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaa 4320 caaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcta.agaaaccatt4380 attatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc 4432 <210> SEQ ID N0:15 <211> LENGTH: 4864 <212> TYPE: DNA
<213> ORGANISM: E. coli <400> SEQ ID N0:15 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300 tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360 ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420 cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540 tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600 tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660 ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720 catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780 cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840 ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900 agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960 tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020 tccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggc 1080 ttcttatgcatgctatactgtttttggcttggggtctatacacccccgcttcctcatgtt 1140 ataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactccc 1200 ctattggtgacgatactttccattactaatccataacatggctctttgccacaactctct 1260 ttattggctatatgccaatacactgtccttcagagactgacacggactctgtatttttac 1320 aggatggggtctcatttattatttacaaattcacatatacaacaccaccgtccccagtgc 1380 ccgcagtttttattaaacataacgtgggatctccacgcgaatctcgggtacgtgttccgg 1440 acatgggctcttctccggtagcggcggagcttctacatccgagccctgctcccatgcctc 1500 cagcgactcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggca 1560 cagcacgatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtc 1620 tgaaaatgagctcggggagcgggcttgcaccgctgacgcatttggaagacttaaggcagc 1680 ggcagaagaagatgcaggcagctgagttgttgtgttctgataagagtcagaggtaactcc 1740 cgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgc 1800 gcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtctttt 1860 ctgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgcc 1920 cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa 1980 atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgg 2040 ggcagcacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg 2100 gctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagc 2160 aggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccc 2220 cactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtac 2280 ttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgg 2340 gaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatg 2400 tgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctg 2460 cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta 2520 tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc 2580 aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag 2640 catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac 2700 caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc 2760 ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgt 2820 aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc 2880 gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga 2940 cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 3000 ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagta 3060 tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga 3120 tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg 3180 cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag 3240 tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc 3300 tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 3360 tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt 3420 cgttcatccatagttgcctgactccggggggggggggcgctgaggtctgcctcgtgaaga 3480 aggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgaggga 3540 gccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctt 3600 tgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaa 3660 agttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgt 3720 tacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaat 3780 ttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaagga 3840 gaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccg 3900 actcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagt 3960 gagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttct 4020 ttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacc 4080 aaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaa 4140 ggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaaca 4200 atattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatc 4260 gcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaaga 4320 ggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacg 4380 ctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatag 4440 attgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagca 4500 tccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcata 4560 acaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattt 4620 ttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccc 4680 cattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt 4740 tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc 4800 taagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccttt 4860 cgtc 4864 <210> SEQ ID N0:16 <211> LENGTH: 4867 <212> TYPE: DNA
<213> ORGANISM: E. coli <400> ID N0:16 SEQ

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60 cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120 ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180 accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240 ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300 tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360 ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420 cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540 tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600 tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660 ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720 catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780 cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840 ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900 agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960 tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020 tccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggc 1080 tcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgcta 1140 taggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccc 1200 tattggtgacgatactttccattactaatccataacatggctctttgccacaactatctc 1260 tattggctatatgccaatactctgtccttcagagactgacacggactctgtatttttaca 1320 ggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcc 1380 cgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccgga 1440 catgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctcc 1500 agcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcac 1560 agcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtct 1620 gaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcg 1680 gcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactccc 1740 gttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcg 1800 cgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttc 1860 tgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgccc 1920 ctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaa 1980 tgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggg 2040 gcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtggg 2100 ctctatggccgctgcggccaggtgctgaagaattgacccggttcctcctgggccagaaag 2160 aagcaggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttcca 2220 gccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaa 2280 gtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaaga 2340 gtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaa 2400 catgtgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactc 2460 gctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacg 2520 gttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaa 2580 ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctga 2640 cgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaag 2700 ataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgct 2760 taccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacg 2820 ctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacc 2880 ccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggt 2940 aagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggta 3000 tgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaac 3060 agtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctc 3120 ttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagat 3180 tacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgc 3240 tcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatctt 3300 cacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagta 3360 aacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct 3420 atttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtga 3480 agaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgag 3540 ggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttg 3600 ctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagc 3660 aaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccag 3720 tgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgc 3780 aatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaa 3840 ggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgatt 3900 ccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatca 3960 agtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatt 4020 tctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatca 4080 accaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgtta 4140 aaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatca 4200 acaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccgggg 4260 atcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcgga 4320 agaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggca 4380 acgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcga 4440 tagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatca 4500 gcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctc 4560 ataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatata 4620 tttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttcccccccc 4680 ccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgt 4740 atttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgac 4800 gtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccc 4860 tttcgtc 4867 <210> SEQ ID N0:17 <211> LENGTH: 78 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:17 gatcaccatg gatgcaatga agagagggct ctgctgtgtg ctgctgctgt gtggagcagt 60 cttcgtttcg cccagcga 78 <210> SEQ ID N0:18 <211> LENGTH: 78 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:18 gatctcgctg ggcgaaacga agactgctcc acacagcagc agcacacagc agagccctct 60 cttcattgca tccatggt 78 <210> SEQ ID N0:19 <211> LENGTH: 27 <212> TYPE: PRT
<213> ORGANISM: Homo sapien <400> SEQ ID N0:19 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser <210> SEQ ID N0:20 <211> LENGTH: 33 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:20 ggtacaaata ttggctattg gccattgcat acg 33 <210> SEQ ID N0:21 <211> LENGTH: 36 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:21 ccacatctcg aggaaccggg tcaattcttc agcacc 36 <210> SEQ ID N0:22 <211> LENGTH: 38 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:22 ggtacagata tcggaaagcc acgttgtgtc tcaaaatc 38 <210> SEQ ID N0:23 <211> LENGTH: 36 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:23 cacatggatc cgtaatgctc tgccagtgtt acaacc 36 <210> SEQ ID N0:24 <211> LENGTH: 39 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:24 ggtacatgat cacgtagaaa agatcaaagg atcttcttg 39 <210> SEQ ID N0:25 <211> LENGTH: 35 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide <400> SEQ ID N0:25 ccacatgtcg acccgtaaaa aggccgcgtt gctgg 35 <210> SEQ ID N0:26 <211> LENGTH: 4864 <212> TYPE: DNA
<213> ORGANISM: E. coli <400> SEQ ID N0:26 tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60 cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120 ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180 accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240 ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300 tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360 ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420 cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480 catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540 tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600 tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660 ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720 catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780 cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840 ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900 agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960 tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020 tccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggc 1080 ttcttatgcatgctatactgtttttggcttggggtctatacacccccgcttcctcatgtt 1140 ataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactccc 1200 ctattggtgacgatactttccattactaatccataacatggctctttgccacaactctct 1260 ttattggctatatgccaatacactgtccttcagagactgacacggactctgtatttttac 1320 aggatggggtctcatttattatttacaaattcacatatacaacaccaccgtccccagtgc 1380 ccgcagtttttattaaacataacgtgggatctccacgcgaatctcgggtacgtgttccgg 1440 acatgggctcttctccggtagcggcggagcttctacatccgagccctgctcccatgcctc 1500 cagcgactcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggca 1560 cagcacgatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtc 1620 tgaaaatgagctcggggagcgggcttgcaccgctgacgcatttggaagacttaaggcagc 1680 ggcagaagaagatgcaggcagctgagttgttgtgttctgataagagtcagaggtaactcc 1740 cgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgc 1800 gcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtctttt 1860 ctgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgcc 1920 cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa 1980 atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgg 2040 ggcagcacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg 2100 gctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagc 2160 aggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccc 2220 cactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtac 2280 ttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgg 2340 gaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatg 2400 tgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctg 2460 cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta 2520 tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc 2580 aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag 2640 catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac 2700 caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc 2760 ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgt 2820 aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc 2880 gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga 2940 cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 3000 ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagta 3060 tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga 3120 tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg 3180 cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag 3240 tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc 3300 tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 3360 tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt 3420 cgttcatccatagttgcctgactccggggggggggggcgctgaggtctgcctcgtgaaga 3480 aggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgaggga 3540 gccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctt 3600 tgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaa 3660 agttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgt 3720 tacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaat 3780 ttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaagga 3840 gaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccg 3900 actcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagt 3960 gagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttct 4020 ttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacc 4080 aaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaa 4140 ggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaaca 4200 atattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatc 4260 gcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaaga 4320 ggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacg 4380 ctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatag 4440 attgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagca 4500 tccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcata 4560 acaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattt 4620 ttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccc 4680 cattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt 4740 tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc 4800 taagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccttt 4860 cgtc 4864 <210> SEQ ID N0:27 <211> LENGTH: 139 <212> TYPE: DNA
<213> ORGANISM:E. coli / HIV-1 <400> SEQ ID N0:27 catgggtctt ttctgcagtc accgtccttg agatctgcca ccatgggcgg caagtggtcc 60 aagaggtccg tgccccaccc cgagtactac aaggactgct aaagcccggg cagatctgct 120 gtgccttcta gttgccagc 139 <210> SEQ ID N0:28 <211> LENGTH: 139 <212> TYPE: DNA
<213> ORGANISM:E. coli / HIV-1 <400> SEQ ID N0:28 catgggtctt ttctgcagtc accgtccttg agatctgcca ccatggccgg caagtggtcc 60 aagaggtccg tgccccaccc cgagtactac aaggactgct aaagcccggg cagatctgct 120 gtgccttcta gttgccagc 139 <210> SEQ ID N0:29 <211> LENGTH: 203 <212> TYPE: DNA
<213> ORGANISM:E. coli / HIV-1 <400> SEQ ID N0:29 catgggtctt ttctgcagtc accgtcctta tatctagatc accatggatg caatgaagag 60 agggctctgc tgtgtgctgc tgctgtgtgg agcagtcttc gtttcgccca gcgagatctc 120 ctccaagagg tccgtgcccc accccgagta ctacaaggac tgctaaagcc cgggcagatc 180 tgctgtgcct tctagttgcc agc 203 <210> SEQ ID N0:30 <211> LENGTH: 651 <212> TYPE: DNA
<213> ORGANISM:Human Immunodificiency Virus - 1 <400> SEQ ID N0:30 atgggtggcaagtggtcaaaacgtagtgtgcctggatggtctactgtaagggaaagaatg 60 agacgagctgagccagcagcagatagggtgagacgaactgagccagcagcagtaggggtg 120 ggagcagtatctcgagacctggaaaaacatggagcaatcacaagtagcaatacagcagct 180 accaatgctgattgtgcctggctagaagcacaagaggatgaggaagtgggttttccagtc 240 agacctcaggtacctttaagaccaatgacttacaagggagctgtagatcttagccacttt 300 ttaaaagaaaaggggggactggaagggctaattcactcacagaaaagacaagatatcctt 360 gatctgtgggtctaccacacacaaggctacttccctgattggcagaactacacaccaggg 420 ccaggaatcagatttccattgacctttggatggtgcttcaagctagtaccagttgagcca 480 gaaaaggtagaagaggccaatgaaggagagaacaactgcttgttacaccctatgagccag 540 catgggatagaggacccggagaaggaagtgttagagtggaggtttgacagcaagctagca 600 tttcatcacgtggcccgagagctgcatccggagtactacaaggactgctga 651

Claims (29)

WHAT IS CLAIMED IS:

1. A pharmaceutically acceptable DNA vaccine, which comprises:
(a) a DNA expression vector; and, (b) a DNA molecule containing a codon optimized open reading frame encoding a Nef protein or immunogenic Nef derivative thereof, wherein upon administration of the DNA vaccine to a host the Nef protein or immunogenic Nef derivative is expressed and generates an immune response which provides a substantial level of protection against HIV-1 infection.

2. A DNA vaccine of claim 1 wherein the DNA molecule encodes wild type Nef.

3. A DNA vaccine of claim 2 wherein the DNA molecule contains the nucleotide sequence as set forth in SEQ ID NO:1.

4. The DNA vaccine of claim 3 which is V1Jns-opt nef (jrfl).

5. A DNA vaccine of claim 2 wherein the DNA molecule expresses a wild type Nef protein which comprises the amino acid sequence as set forth in SEQ
ID NO:2.

6. A DNA vaccine of claim 1 wherein the DNA molecule encodes an immunogenic Nef derivative which contains a nucleotide sequence encoding a leader peptide.

7. A DNA vaccine of claim 6 wherein the DNA molecule encodes an immunogenic Nef derivative which contains a nucleotide sequence encoding a human tissue plasminogen activator leader peptide.

8. A DNA vaccine of claim 7 wherein the DNA molecule contains the nucleotide sequence as set forth in SEQ 1D NO:3.

9. The DNA vaccine of claim 8 which is V1Jns-opt tpanef.

10. A DNA vaccine of claim 7 wherein the DNA molecule expresses an immunogenic Nef derivative which comprises the amino acid sequence as set forth in SEQ ID NO:4.

11. A DNA vaccine of claim 6 wherein the DNA molecule encodes an immunogenic Nef derivative modified at the dileucine motif of amino acid residue 174 and amino acid residue 175.

12. A DNA vaccine of claim 11 wherein the DNA molecule encodes an immunogenic Nef derivative which contains a nucleotide sequence encoding a human tissue plasminogen activator leader peptide.

13. A DNA vaccine of claim 12 wherein the DNA molecule contains the nucleotide sequence as set forth in SEQ ID NO:7.

14. The DNA vaccine of claim 13 which is V1Jns-opt tpanef (LLAA).

15. A DNA vaccine of claim 11 wherein the DNA molecule expresses an immunogenic Nef derivative which comprises the amino acid sequence as set forth in SEQ ID NO:8.

16. A DNA vaccine of claim 11 wherein the DNA molecule encodes a Nef protein where the glycine residue of amino acid residue 2 of Nef is modified to encode for an amino acid residue other the glycine.

17. A DNA vaccine of claim 16 wherein the DNA molecule contains the nucleotide sequence as set forth in SEQ ID NO:5.

18. A DNA vaccine of claim 17 which is V1Jns-opt nef (G2A LLAA).

19. A DNA vaccine of claim 16 wherein the DNA molecule expresses an immunogenic Nef derivative which comprises the amino acid sequence as set forth in SEQ ID NO:6.

20. A DNA vaccine of claim 1 which further comprises an adjuvant.

21. A DNA vaccine of claim 20 wherein the adjuvant is selected from the group consisting of alumunum phosphate, calcium phosphate and a non-ionic block copolymer.

22. A pharmaceutically acceptable DNA vaccine, which comprises:
(a) a DNA expression vector; and, (b) a DNA molecule containing an open reading frame encoding a Nef protein or immunogenic Nef derivative thereof, wherein upon administration of the DNA
vaccine to a host the Nef protein or immunogenic Nef derivative is expressed and generates an immune response which provides a substantial level of protection against HIV-1 infection.

23. The DNA vaccine of claim 22wherein the DNA molecule expresses a wild type Nef protein which comprises the amino acid sequence as set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.

24. A DNA vaccine of claim 22 which further comprises an adjuvant.

25. A DNA vaccine of claim 23 whrerein the adjuvant is selected from the group consisting of alumunum phosphate, calcium phosphate and a non-ionic block copolymer.

26. A method for inducing a cell mediated immune (CTL) response against infection or disease caused by virulent strains of HIV which comprises administering into the tissue of a vertebrate host a pharmaceutically acceptable DNA vaccine composition which comprises a DNA expression vector and a DNA molecule containing a codon optimized open reading frame encoding a Nef protein or immunogenic Nef derivative thereof, wherein upon administration of the DNA
vaccine to the vertebrate host the Nef protein or immunogenic Nef derivative is expressed and generates the cell-mediated immune (CTL) response.

27. The method of claim 26 wherein the vertebrate host is a human.

28. The method of claim 26 wherein the DNA vaccine is selected from the group consisting of V1Jns-opt nef (jrfl), V1Jns-opt tpanef, V1Jns-opt tpanef (LLAA), and V1Jns-opt nef (G2A LLAA).

29. A substantially purified protein which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.