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WO1999003888A1 - Recepteurs de structure variable associes a une incursion retrovirale dans des cellules - Google Patents

Recepteurs de structure variable associes a une incursion retrovirale dans des cellules Download PDF

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Publication number
WO1999003888A1
WO1999003888A1 PCT/US1998/014857 US9814857W WO9903888A1 WO 1999003888 A1 WO1999003888 A1 WO 1999003888A1 US 9814857 W US9814857 W US 9814857W WO 9903888 A1 WO9903888 A1 WO 9903888A1
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WO
WIPO (PCT)
Prior art keywords
human
cell
promoting agent
translocation promoting
bonzo
Prior art date
Application number
PCT/US1998/014857
Other languages
English (en)
Inventor
Dan R. Littman
Hongkui Deng
Derya Unutmaz
Vineet N. Kewalramani
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New York University
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Filing date
Publication date
Application filed by New York University filed Critical New York University
Publication of WO1999003888A1 publication Critical patent/WO1999003888A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines

Definitions

  • the present invention relates generally to the infection of target cells by HIN-1, HJN- 2, and SIN and more particularly to agents identified herein that mediate the entry of retroviruses into such target cells, and to the diagnostic and therapeutic uses to which such agents may be put.
  • the human immunodeficiency viruses infect CD4 + macrophages and T helper cells.
  • HIV- 1 entry requires cell surface expression of CD4, to which the viral envelope glycoproteins bind, several studies have suggested that it is not sufficient for fusion of the viral envelope to the cellular plasma membrane.
  • early studies have shown that while human cells expressing a transfected CD4 gene were permissive for virus entry, murine cells expressing human CD4 were not. These findings led to the suggestion that there is a species-specific cell surface cofactor required in addition to CD4 for HIN-1 entry.
  • T- tropic strains both M-tropic and T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4 [Chew et al., J. Virol, 71:2705-2714 (1997); Marcon et al., J. Virol, 71:2522-2527 (1997); and Edinger et al., Proc. Natl. Acad. Sci. USA, 94:4005-4010 (1997)]. More importantly, SIV strains were also found to infect CD4 + cells that lack CCR5 [Chen et al., 1997, supra; and Edinger et al., 1997, supra].
  • CCR5 -tropic viruses are primarily involved in transmission, while viruses with broader tropism, particularly for CXCR4, emerge during progression to immunodeficiency [Fauci, Nature, 384:529-534 (1996)]. It is not yet known whether appearance of CXCR4-tropic viruses is a consequence or the cause of immune system decline. Insight into this key problem of virus evolution is likely to require experimental manipulation in animal models. Infection of non-human primates with SIV is currently the only good animal model for studying pathogenesis of the immunodeficiency viruses [Desrosiers, Annu Rev Immunol, 8:557-578 (1990)]. Moreover, different species of non-human primates vary widely in their responses to SIV infection.
  • translocation promoting agents other than CCR5 and CXCR4 that function in conjunction with CD4 during SIV and/or HIV infection of human cells as well in the subsequent disease progression. Further, there is a need to determine the specific strains of the retroviruses that can use such translocation promoting agents as alternatives to CXCR4 and CCR5. In addition, there is a need to provide methods of identifying drugs that can interfere with retroviral infection by hindering the interaction of CD4, the various translocation promoting agents and the retroviral envelope glycoproteins. The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
  • the present invention provides an expression cloning strategy to identify SIV receptors which may play a role in human acquired immunodeficiency disease.
  • Two particular nucleic acids encoding two members of the 7-transmembrane G-protein coupled receptor family were identified and isolated by the methods described herein. These SIV receptors were also found to be used by particular strains of HIV-2 and M- tropic HIV-l.
  • One aspect of the present invention provides an isolated nucleic acid encoding a translocation promoting agent that is substantially homologous to SEQ ID NO: 1.
  • the nucleic acid is obtained from a primate.
  • the nucleic acid encodes a translocation promoting agent that is substantially homologous to SEQ ID NO:3.
  • the isolated nucleic acid encodes a translocation promoting agent that is substantially homologous to SEQ ID NO:5.
  • the isolated nucleic acid encodes a translocation promoting agent isolated from a human source.
  • the present invention also includes nucleic acid primers and/or probes for all of the nucleic acids of the present invention.
  • the nucleic acids of the present invention may be RNA, or single or doubled stranded DNA, including recombinant DNA.
  • the present invention also includes an isolated nucleic acid which encodes a translocation promoting agent having the amino acid sequence of SEQ ID NO:2 having one or more conservative substitutions.
  • the isolated nucleic acid encodes a translocating promoting agent having the amino acid sequence of SEQ ID NO:2.
  • the isolated nucleic acid has a nucleic acid sequence of SEQ ID NO:l.
  • the present invention also includes an isolated nucleic acid encoding a translocation promoting agent having the amino acid sequence of SEQ ID NO:4 having one or more conservative substitutions.
  • the isolated nucleic acid encodes a translocation promoting agent having the amino acid sequence of SEQ ID NO:4.
  • the isolated nucleic acid has the nucleic acid sequence of SEQ ID NO:3.
  • the present invention also includes an isolated nucleic acid encoding a translocation promoting agent having the amino acid sequence of SEQ ID NO:6 having one or more conservative substitutions.
  • the isolated nucleic acid encodes a translocating promoting agent having the amino acid sequence of SEQ ID NO:6.
  • the isolated nucleic acid has the nucleic acid sequence of SEQ ID NO:5.
  • the present invention also includes an isolated nucleic acid encoding a translocation promoting agent having the amino acid sequence of SEQ ID NO: 10 having one or more conservative substitutions.
  • the isolated nucleic acid encodes a translocating promoting agent having the amino acid sequence of SEQ ID NO:10.
  • the isolated nucleic acid has the nucleic acid sequence of SEQ ID NO:9.
  • the present invention also includes an isolated nucleic acid encoding a translocation promoting agent having the amino acid sequence of SEQ ID NO: 12 having one or more conservative substitutions.
  • the isolated nucleic acid encodes a translocating promoting agent having the amino acid sequence of SEQ LD NO: 12.
  • the isolated nucleic acid has the nucleic acid sequence of SEQ ID NO: 11.
  • the present invention also includes nucleic acids which contain 18 or more nucleotides that hybridize to a nucleic acid having the nucleotide sequence of SEQ ID NO:l under standard conditions.
  • the present invention also includes nucleic acids which contain 18 or more nucleotides that hybridize to a nucleic acid having the nucleotide sequence of SEQ ID NO: 3 under standard conditions.
  • the present invention also includes nucleic acids which contain 18 or more nucleotides that hybridize to a nucleic acid having the nucleotide sequence of SEQ ID NO:5 under standard conditions.
  • the present invention also includes nucleic acids which contain 18 or more nucleotides that hybridize to a nucleic acid having the nucleotide sequence of SEQ ID NO: 9 under standard conditions.
  • the present invention also includes nucleic acids which contain 18 or more nucleotides that hybridize to a nucleic acid having the nucleotide sequence of SEQ ID NO: 11 under standard conditions.
  • the present invention also provides nucleic acids encoding fusion proteins containing the translocation promoting agents of the present invention and a peptide tag or protein.
  • fusion proteins can include, for example Bonzo fused with green fluorescent protein, or Bonzo fused with glutathione-s-transferase.
  • DNA constructs comprising the isolated nucleic acids of the present invention, that are operatively linked to an expression control sequence.
  • the nucleic acid has the nucleotide sequence of SEQ ID NO: 1.
  • the present invention also includes methods of using the DNA constructs of the present invention to express the translocation promoting agent which they encode.
  • One such embodiment comprises introducing the construct into a host cell and expressing the translocation promoting agent in that host cell.
  • the method includes purifying the translocation promoting agent that was expressed.
  • the present invention also includes unicellular host cells transformed with the DNA constructs of the present invention.
  • the unicellular host is a primate cell.
  • the unicellular host is a human cell.
  • translocation promoting agent having the amino acid sequence that is substantially homologous to SEQ ID NO:2.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:2 having one or more conservative substitutions.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:2.
  • translocation promoting agent having the amino acid sequence that is substantially homologous to SEQ ID NO:4
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:4 having one or more conservative substitutions.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:4.
  • translocation promoting agent having the amino acid sequence that is substantially homologous to SEQ ID NO:6.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:6 having one or more conservative substitutions.
  • the translocation promoting agent has the amino acid sequence ofSEQ ID NO:6.
  • translocation promoting agent having the amino acid sequence that is substantially homologous to SEQ ID NO: 10.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO: 10 having one or more conservative substitutions.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:10.
  • SUBSTTTuTESHEET(RULE26) Another aspect of the present invention provides an isolated translocation promoting agent having the amino acid sequence that is substantially homologous to SEQ ID NO: 12.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO: 12 having one or more conservative substitutions.
  • the translocation promoting agent has the amino acid sequence of SEQ ID NO:12.
  • Peptide fragments of all the translocation promoting agents of the present invention are also part of the present invention. Such fragments can be generated by treatment with proteolytic enzymes and the like.
  • the present invention also provides antibodies to the translocation promoting agents of the present invention.
  • the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO:2 having one or more conservative substitutions.
  • the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO:2.
  • the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO:4 having one or more conservative substitutions.
  • the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO:4.
  • the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO: 6 having one or more conservative substitutions.
  • the antibody is to the translocation promoting agent having the amino acid sequence ofSEQ ID NO:6. In yet another embodiment of this type the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO: 10 having one or more conservative substitutions. In a preferred embodiment of this type the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO: 10. In still another embodiment of this type the antibody is to the translocation promoting agent having the amino acid sequence of SEQ ID NO: 12 having one or more conservative substitutions. In a preferred embodiment of this type the antibody is to the translocation promoting agent having the amino acid sequence of SEQ LD NO:12.
  • the antibody is a monoclonal antibody and/or a chimeric antibody.
  • the present invention also includes an immortal cell line that produces a monoclonal antibody of the present invention.
  • the present invention also provides a mammalian cell that expresses human CD4 and is transfected with a vector encoding Bonzo.
  • Bonzo is a human Bonzo.
  • the human Bonzo has an amino acid sequence of SEQ ID NO:2.
  • a mammalian cell which expresses CD4, but expresses low levels or no CXCR4 and CCR5, in the absence of transduction with a vector encoding CXCR4 and/or CCR5, is transfected with a vector encoding human Bonzo.
  • the mammalian cell further comprises a vector encoding one or more translocation promoting agents such as CCR5, CXCR4, CCR2b, CCR3, and BOB.
  • a related aspect of the invention is a mammalian cell that is transfected with a vector encoding human CD4 and a vector encoding human or primate BOB.
  • the primate BOB has the amino acid sequence of SEQ ID NO: 10 or 12.
  • the human BOB has the amino acid sequence of SEQ ID NO:8.
  • a mammalian cell which expresses CD4, but expresses low levels or no CXCR4 and CCR5, in the absence of transduction with a vector encoding CXCR4 and/or CCR5, is transfected with a vector encoding human BOB.
  • the mammalian cell further comprises a vector encoding one or more translocation promoting agents such as CCR5, CXCR4, CCR2b, CCR3, and Bonzo.
  • the present invention also includes the mammalian cells of the present invention attached to solid support matrices.
  • the mammalian cell is a human cell.
  • transgenic non-human mammal comprising a DNA construct containing a human CD4 gene and DNA construct containing a human translocation promoting agent such as BOB or Bonzo.
  • the transgenic non-human mammal further comprises one or more DNA constructs which contain an alternative human translocation promoting agent such as CCR5, CXCR4, CCR2b, and/or CCR3.
  • the transgenic non-human mammal is a mouse.
  • the present invention further provides a method of identifying a nucleic acid that encodes a human translocation promoting agent which in conjunction with CD4 serves as a receptor for the entry into a cell of a virus having a specific viral envelope glycoprotein.
  • One such method includes transfecting a mammalian cell with a viral vector containing a human cDNA with a flanking- viral insert.
  • the mammalian cell expresses human CD4 and a known human translocation promoting agent, such as CCR5, but does not express the human translocation promoting agent encoded by the human cDNA which is obtained from a human cDNA library.
  • the mammalian cell is transfected with a first selectable viral vector pseudotyped with the specific viral envelope glycoprotein.
  • the first selectable viral vector encodes a first selectable marker.
  • the mammalian cell is then identified by expressing the first selectable marker.
  • the identified mammalian cell is next transfected with a second selectable viral vector that is pseudotyped with a particular viral envelope glycoprotein.
  • the second selectable viral vector encodes a second selectable marker, and the known human translocation promoting agent can serve in conjunction with CD4 as a receptor for entry of a virus having the particular envelope glycoprotein.
  • An identified mammalian cell is then selected which does not express the second selectable marker.
  • DNA is then extracted from the selected mammalian cell and amplified by PCR using primers for the flanking insert from the viral vectors. In this way a nucleic acid encoding a human translocation promoting agent that can serve in conjunction with CD4 as a receptor for entry of virus having the specific viral
  • suBs ⁇ ruTE SHEET (RULE 26) envelope glycoprotein is identified.
  • the first selectable viral vector is a selectable replication- defective virus.
  • the first selectable viral vector is an HIV-pseudotyped vector.
  • the second selectable viral vector is a selectable replication-defective virus.
  • the second selectable viral vector is an HIN-pseudotyped vector.
  • both the first selectable viral vector and the second selectable viral vector are HJN-pseudotyped vectors.
  • the viral vector containing the human cD ⁇ A is obtained from the supernatant of BOSC23 packaging cells after subcloning the human cD ⁇ A library in the retrovirus pMX and transfecting the human cD ⁇ A library into the BOSC23 packaging cells.
  • the mammalian cell is 3T3.CD4.
  • the first selectable marker confers antibiotic resistance to the cell.
  • the antibiotic is puromycin and the identification of the mammalian cell expressing the first selectable marker is performed by administering puromycin to the cells and identifying cells that are puromycin resistant.
  • the first selectable marker is green fluorescent protein.
  • the second selectable marker is green fluorescent protein.
  • the first selectable marker is luciferase.
  • the second selectable marker is luciferase.
  • the known translocation promoting agent used in these methods can be any translocating promoting agent known including CCR5, CXCR4, CCR2b, CCR3, Bonzo, and BOB.
  • the known translocation promoting agent is CCR5 and the particular viral envelope glycoprotein is JRFL.
  • the present invention also includes assays for selecting for a suspected therapeutic agent for possible use in the treatment of AIDS with the use of one of the cells of the present invention.
  • the cell is a mammalian cell that expresses human CD4 and is transfected with a vector encoding human Bonzo.
  • the mammalian cell is transfected with a vector encoding human CD4 and a vector encoding a human translocation promoting agent such as Bonzo or BOB.
  • Bonzo and BOB are human Bonzo and human BOB.
  • One such method for selecting a suspected therapeutic agent for possible use in the treatment of AIDS comprises administering a potential therapeutic agent to the cell.
  • the cell is next infected with a virus pseudotyped with an HIV envelope glycoprotein. This is followed by measuring the ability of the cell to resist the infection.
  • the potential therapeutic agent is selected when the measured ability of the cell to resist the infection is statistically greater in the presence of the potential therapeutic agent than in the absence of the potential therapeutic agent.
  • the selected potential therapeutic agent is a suspected therapeutic agent.
  • the virus contains a marker protein and measuring the ability of the cell to resist the infection is performed by detecting the amount of marker protein expressed in the cell. In this case the measured ability of the cell to resist the infection is inversely proportional to the amount of marker protein detected.
  • the marker protein is either luciferase or green fluorescent protein.
  • the present invention also includes an assay for selecting a plausible therapeutic agent for possible use in the treatment of AIDS with the use of a transgenic non-human mammal that comprises a DNA construct containing a human CD4 gene and a DNA construct containing either human Bonzo or human BOB.
  • One such method comprises administering a suspected therapeutic agent to the transgenic non-human mammal. Then the transgenic non-human mammal is infected with a virus pseudotyped with an HIV envelope glycoprotein. The ability of the transgenic non- human mammal to resist infection is then measured. The suspected therapeutic agent is selected when the measured ability of the transgenic non-human mammal to resist the infection is statistically greater in the presence of the suspected therapeutic agent than in the absence of the therapeutic agent. In this case the selected therapeutic agent is a plausible therapeutic agent.
  • the present invention also includes a method of filtering a biological fluid to remove and/or isolate a virus expressing an HIV envelope glycoprotein that binds with CD4 and Bonzo and/or BOB wherein the biological fluid is passed through a mammalian cell which expresses human CD4 and human Bonzo and/or human BOB and is attached to a solid support matrix.
  • the mammalian cell is transfected with a vector encoding human CD4 and a vector encoding human Bonzo and/or a vector encoding human BOB.
  • a method for identifying a ligand for human Bonzo In one such assay a potential ligand is contacted with a mammalian cell that expresses human Bonzo and CD4, but the mammalian cell does not express CCR5, CXCR4, CCR2b, CCR3, and BOB. Next the mammalian cell is transfected with a selectable replication defective virus (such as HIV) pseudotyped with a specific viral envelope glycoprotein. In this case Bonzo in conjunction with CD4 serves as a receptor for entry into a cell of a virus having the specific viral envelope glycoprotein.
  • a selectable replication defective virus such as HIV
  • the marker protein is detected and a ligand is selected when the amount of marker protein detected is less than that detected when the transfection of the mammalian cells with the selectable replication-defective virus is performed without the prior contacting of a potential ligand with the mammalian cell.
  • the marker protein is either luciferase or green fluorescent protein.
  • the method includes contacting the selected ligand with purified human Bonzo and then detecting the binding of that ligand to the purified human Bonzo.
  • a ligand is identified by its binding to the purified human Bonzo.
  • the ligand binds to the purified human Bonzo under standard physiological conditions (e.g. lOOmM NaCl pH7.4, at 37EC) and has a KD of less than 10 "6 Molar.
  • a method for identifying a ligand for human BOB.
  • a potential ligand is contacted with a mammalian cell that expresses human BOB and CD4, but the mammalian cell does not express CCR5, CXCR4, CCR2b, CCR3, and Bonzo.
  • the mammalian cell is transfected with a selectable replication defective virus (such as HIV) pseudotyped with a specific viral envelope glycoprotein.
  • a selectable replication defective virus such as HIV
  • BOB in conjunction with CD4 serves as a receptor for entry into a cell of a virus having the specific viral envelope glycoprotein.
  • the marker protein is detected and a ligand is selected when the amount of marker protein detected is less than that detected when the transfection of the mammalian cells with the selectable replication-defective virus is performed without the prior contacting of a potential ligand with the mammalian cell.
  • the marker protein is either luciferase or green fluorescent protein.
  • the method includes contacting the selected ligand which was selected with purified human BOB and then detecting the binding of that ligand to the purified human BOB.
  • a ligand is identified by binding to the purified human BOB.
  • the ligand binds to the purified human BOB under standard physiological conditions (e.g. lOOmM NaCl pH7.4, at 37EC) and has a KD of less than 10 "6 Molar.
  • FIGURE 1 Specificity of SIV Envs for chemokine receptors on target cells.
  • Figure la depicts the results of the infection of 3T3.CD4 cells stably expressing CCR1, CCR5, or CXCR4 with luciferase reporter viruses pseudotyped with Envs of HTV-l (HXB2 and JRFL), SIV (macl Al 1 and agmTYOl) or VSV-G.
  • Figure lb depicts the infection of U87 cells and U87.CD4 cells with the pseudotyped reporter viruses.
  • Figure lc depicts the results of the infection of transformed CD4 + cell lines with the pseudotyped reporter viruses. Ly sates were assayed 3 days post-infection for luciferase activity as described in Methods (Example 1).
  • FIGURE 2 Expression cloning of novel SIV receptors.
  • Figure 2a shows an outline of the expression cloning strategy used to isolate SIV receptors.
  • Figure 2b depicts the results of the infection of representative 3T3.CD4 clones, previously selected with H ⁇ V-puro(SIV) pseudotyped virus, with HTV-luc pseudotyped with SIV and M-tropic HIV-1 Envs.
  • Figure 2c shows the amino acid sequence alignment of the receptors encoded by the BOB and Bonzo cDNAs with HIV receptors CXCR4 and CCR5. Identical amino acids are shaded and the putative extracellular domains (based on CCR5 alignment) are boxed.
  • the "DRY box” sequence which is involved in signaling and is characteristic of chemokine receptors, is highlighted in bold. Bonzo contains a divergent "DRY box” motif compared to other HIV receptors; this sequence has thus been observed in only one other chemokine receptor, EBI 2.
  • FIGURE 3 BOB and Bonzo mediate entry of SIV, H ⁇ V-2 and M-tropic or dual-tropic HIV-1 strains.
  • Figure 3a depicts the results of the infection of 293T cells transiently cotransfected with plasmids encoding CD4 plus CCR1, Bonzo, BOB, or CCR5. The cells were infected 2 days later with luciferase reporter viruses (e.g. HIN- luc) pseudotyped with different SIN or fflV-2 Envs.
  • the HIV-2 Envs, UC 1 , FO784, ST/SXB1 and 7312a have been shown to use CCR5; ST/24.1 and ROD can use both CCR5 and CXCR4; UC2 is known to use only CXCR4.
  • Figure 3b depicts the results of the infection of the transiently transfected 293T cells with HIV-luc pseudotyped with HIN-1 Envs or control VSV-G.
  • Target cells expressing CXCR4 were also included to identify T-tropic Envs, even though 293T cells express endogenous CXCR4.
  • Figure 3c depicts the results of 3T3.CD4 cells stably expressing CCR1, BOB, Bonzo, CCR5 or CXCR4 which were infected with the luciferase reporter viruses as described above. Luciferase activity was measured on day 3. Similar results were obtained in three independent experiments.
  • FIGURE 4 Expression of BOB and Bonzo mR ⁇ As in human lymphoid tissues and cell lines.
  • Figure 4a shows an R ⁇ A tissue blot of various human tissues (Clontech) probed with full length BOB and Bonzo cD ⁇ As as described in Methods (Example 1). The signals correspond to 2.1 kb Bonzo and 2.2 Kb BOB transcripts.
  • Figure 4b depicts the RT-PCR analysis of BOB and Bonzo transcripts in human PBMC, resting or stimulated with PHA (Sigma) for 7 days, and purified lymphocyte subsets.
  • the present invention provides two new HIV/SIV translocation promoting agents, Bonzo and BOB.
  • Such translocation promoting agents act in conjunction with CD4 to serve as a receptor for the entry into a cell of a virus having a specific viral envelope glycoprotein.
  • the use of these new receptors in the experimental infection of nonhuman primates with particular SIV and HIV strains can provide important insights into both the viral transmission and mechanisms of SIV and HIV induced acquired immune deficiency syndrome.
  • the ability of HIVs to infect cells by way of these newly disclosed receptors must now be included in the strategies aimed at blocking CCR5-tropic virus entry.
  • the present invention provides a facile method of identifying still other such translocation promoting agents (see Example 1).
  • SUBSmUTE SHEET (RULE 26) variables that influence the outcome of SIV infection. Therefore it is important to determine the correlation between the ability of primary strains of SIV to use the receptors of the present invention and the susceptibility of the animals to disease progression.
  • the present invention provides animal model systems, developed from the teachings herein, for studying HIV infection and pathogenesis. This allows testing of drugs in an animal system prior to human trials. This discovery allows identification of additional related G-protein coupled receptors that have a role in the broadening of the viral host range in vivo and in pathogenesis in organ systems such as the brain.
  • chemokine receptors encoded by other viruses may serve to broaden the host range of HIV in individuals infected with both HIV and such viruses. This can therefore increase the range of tissues infected or provide a ligand for HIN envelope that can result in deleterious signal transduction in various tissues. This information could lead to novel approaches to block the synergy between HIV and viral cofactors.
  • translocation promoting agent is used herein interchangeably with the terms “translocating promoter”, “translocating promoting agent” and “translocating promoting protein”, and refer to receptor proteins found on, or in membranes of CD4 + cells that interact with and/or in conjunction with CD4 in HIV and/or SIV translocation into the cell.
  • Two translocation promoting agents exemplified in the present invention are the proteins named "Bonzo” and "BOB".
  • Bonzo is a human protein having an amino acid sequence of SEQ ID NO:2.
  • Bonzo is an African green monkey protein having an amino acid sequence of SEQ ID NO:4.
  • Bonzo is a pigtail macaque protein having an amino acid sequence of SEQ ID NO:6.
  • the translocation promoting agent is "BOB", a human protein having an amino acid sequence of SEQ ID NO:8.
  • BOB a human protein having an amino acid sequence of SEQ ID NO:8.
  • a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.
  • a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.
  • the term "about” means within 20%, preferably within 10%, and more preferably within 5%.
  • the present invention contemplates isolation of a gene encoding a translocation promoting agent of the invention, including a full length, or naturally occurring form of translocation promoting agent, and any antigenic fragments thereof from any animal, particularly mammalian and more particularly human source.
  • a gene refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.
  • conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature.
  • a “vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a cell has been "transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.
  • the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a "nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5N to 3N direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra).
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55E, can be used, e.g., 5x SSC, 0.1 % SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC,
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40%o formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • RNA:RNA, DNA:RNA, DNA:DNA The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al., supra, 9.50-0.51).
  • the position of mismatches becomes more important, and the length of the ohgonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).
  • a minimum length for a hybridizable nucleic acid is at least about 18 nucleotides; preferably at least about 36 nucleotides; and more preferably the length is at least about 48 nucleotides.
  • nucleic acids can be used as primers or nucleic acid probes for the nucleic acids encoding the translocation promoting agents of the present invention.
  • standard hybridization conditions refers to a T m of 55EC, and utilizes conditions as set forth above.
  • the T m is 60EC; in a more preferred embodiment, the T m is 65EC.
  • Homologous recombination refers to one method of inserting a foreign DNA sequence of a vector into chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5N (amino) terminus and a translation stop codon at the 3N (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3N to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3N direction) coding sequence.
  • the promoter sequence is bounded at its 3N terminus by the transcription initiation site and extends upstream (5N direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • a “signal sequence” is included at the beginning of the coding sequence of a protein to be secreted or expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
  • the term "translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.
  • sequence homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667).
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that do not share a common evolutionary origin (see Reeck et al., supra).
  • sequence similarity when modified with an adverb such as “substantially,” may refer to sequence similarity and not a common evolutionary origin.
  • two DNA sequences are "substantially homologous" or “substantially similar” when at least 50% (preferably at least 75%, and most preferably at least 90 to 95%) of the nucleotides match over the defined length of the DNA coding sequences.
  • Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 30% of the amino acids are identical, or greater than about 60% are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program.
  • corresponding to is used herein to refer similar or homologous sequences, more preferably substantially similar or substantially homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • corresponding to refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
  • a gene encoding a translocation promoting agent can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining a particular translocating promoting agent gene are well known in the art, as described above (see, e.g., Sambrook et al., 1989, supra).
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., a macrophage/monocyte or T lymphocyte cDNA library, since these are the cells that evidence highest levels of expression of translocation promoting proteins), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • identification of the specific DNA fragment containing the desired translocation promoting agent gene may be accomplished in a number of ways. For example, if an amount of a portion of a translocation promoting agent gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).
  • a set of oligonucleotides corresponding to the partial amino acid sequence information obtained for the translocation promoting agent protein can be prepared and used as probes for DNA encoding the translocation promoting agent, or as primers for cDNA or mRNA (e.g., in combination with a poly-T primer for RT-PCR).
  • a fragment is selected that is highly unique to the translocation promoting agent of the invention. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In a specific embodiment, high stringency hybridization conditions are used to identify a homologous translocation promoting agent gene.
  • the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, or partial amino acid sequence of a translocation promoting agent as disclosed herein.
  • the presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
  • cDNA clones or DNA clones which hybrid-select the proper rnRNAs, can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for a translocation promoting agent of the present invention.
  • polyclonal antibody to the human translocation promoting agent can be used to confirm expression of the translocation promoting agent, (either simian or human).
  • Receptors that act as translocation promoting agents for particular HTV or SIV strains can be identified using an HTV-luciferase vector (for example) pseudotyped with the corresponding HTV or STV envelope glycoproteins (ENVS) to infect CD4 + cell lines.
  • the genes encoding such identified translocation promoting agents can then be isolated.
  • a human T cell clone cDNA library can be subcloned into a viral vector, preferably a retroviral vector, e.g., pMX, and then transfected into a packaging cell such as BOSC23. The supernatant from these cells can then be used to transfect the CD4 + cells (e.g., 3T3.CD4 cells).
  • the transduced cells are next challenged with a selectable replication-defective virus, such as HTV-puro, (which bestows puromycin resistance to the transfected cell) pseudotyped with virus bearing ENVS from HIV or SIV strains, for example STV agmTY01 or SIV maclA11 .
  • the cells are next treated with puromycin to select for cells infected by the HTV-puro virus (which bestows the puromycin resistance).
  • the puromycin resistant colonies are next transfected with an HIV pseudotyped with an HTV envelope glycoprotein which has a specific requirement for a known translocation promoting agent.
  • the genomic DNA is next extracted from the puromycin resistant colonies that were also resistant to the infection of the HTV pseudotyped with an HIV envelope glycoprotein which has the specific requirement for a known translocation promoting agent. In the case in which the virus was pseudotyped with JRFL, the cells in these colonies would be lacking CCR5.
  • the extracted DNA is then subjected to PCR amplification using the primer flanking inserts from the viral vectors. Nucleic acids which encode the translocation promoting agents are detected in this manner.
  • the PCR products can then be subcloned into an expression vector and the DNA sequences can then be determined.
  • the present invention also relates to genes encoding analogs and derivatives of the translocation promoting agent of the invention, that have the same or homologous functional activity as the translocation promoting agent, and homologs thereof from other species.
  • the production and use of derivatives and analogs related to the translocation promoting agent are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type translocation promoting agent of the invention.
  • the translocation promoting agent contains a different cytoplasmic domain, e.g., which associates the protein with the cell membrane but cannot mediate G protein activation and/or translocation.
  • translocation promoting agents can be made by altering nucleic acids encoding translocation promoting agents by substitutions, additions or deletions that provide for functionally modified molecules.
  • derivatives are made that have diminished HIV-translocational activity relative to the native translocating promoting agent.
  • Other derivatives may encode soluble fragments of the translocating promoting agent extracellular domain that have the lower affinity for the natural ligand of the translocating promoting agent of the invention but greater affinity for an HIV or STV envelope protein.
  • Such soluble derivatives may be potent inhibitors of HTV or SIV binding to the translocation promoting agent on cells, e.g., to Bonzo or BOB on macrophages and T cells.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as a translocation promoting agent gene may be used in the practice of the present invention.
  • DNA sequences which encode substantially the same amino acid sequence as a translocation promoting agent gene may be used in the practice of the present invention.
  • These include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of translocation promoting agent genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • the traiislocation promoting agent derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a translocation promoting agent protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in one or more conservative amino acid substitutions.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. Such substitutions are defined herein as conservative substitutions.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.
  • substitutions are: - Lys for Arg and vice versa such that a positive charge may be maintained;
  • translocation promoting agent derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned translocation promoting agent gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the translocation promoting agent-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutations diminish the HTV translocation activity of the mutated translocation promoting agent gene product.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site- directed mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 ⁇ plasmid.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.
  • the nucleotide sequence coding for a translocation promoting agent of the present invention, or antigenic fragment, derivative or analog thereof, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter.”
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • promoter a promoter in an expression vector of the invention.
  • Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding a translocation promoting agent and/or its flanking regions.
  • potential chimeric partners for a translocation promoting agent include other transmembrane domains, or a domain for modification with a phospholipid anchor.
  • Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA e.g., bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant translocation promoting agent of the invention, or fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • the cell into which the recombinant vector comprising the nucleic acid encoding a translocation promoting agent is cultured in an appropriate cell culture medium under conditions that provide for expression of translocation promoting agent by the cell.
  • Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
  • translocation promoting agent may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • the translocation promoting agent is under the control of the CD4 enhancer/promoter/silencer, as described [Killeen et al., (1993) supra].
  • Promoters which may be used to control the translocation promoting agent gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3N long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
  • SUBSTTTUTE SHEET (RULE 26) 318:533-538; Alexander et al, 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha- fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.
  • alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of S V40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El, pCRl , ⁇ BR322, ⁇ Mal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage 8, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • phage DNAS e.g., the numerous derivatives of phage 8, e.g.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage [e.g., of signal sequence]) of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system can be used to produce an nonglycosylated core protein product.
  • the transmembrane translocation promoting agent expressed in bacteria may not be properly folded.
  • Expression in yeast can produce a glycosylated product.
  • Expression in eukaryotic cells can increase the likelihood of "native" glycosylation and folding of a heterologous protein.
  • expression in mammalian cells can provide a tool for reconstituting, or constituting, translocation promoting activity.
  • different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.
  • Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dexfran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • a recombinant translocation promoting agent expressed as an integral membrane protein can be isolated and purified by standard methods.
  • the integral membrane protein can be obtained by lysing the membrane with detergents, such as but not limited to, sodium dodecyl sulfate (SDS), Triton X-100, nonidet P-40 (NP- 40), digoxin, sodium deoxycholate, and the like, including mixtures thereof. Solubilization can be enhanced by sonication of the suspension. Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing inclusion bodies, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above.
  • detergents such as but not limited to, sodium dodecyl sulfate (SDS), Triton X-100, nonidet P-40 (NP- 40), digoxin, sodium deoxycholate, and the like, including mixtures thereof. Solubilization can be enhanced by sonication of the suspension. Solub
  • the solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
  • PAGE polyacrylamide gel electrophoresis
  • isoelectric focusing e.g., isoelectric focusing
  • 2-dimensional gel electrophoresis e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
  • the gene encoding the protein is transfected or used to transform host cells. More preferably, such host cells are transfected to co-express human CD4, and more preferably, such cells lack the ability to express an endogenous or native translocation promoting agent. Co-expression of the translocation promoting agent and CD4 facilitates HTV and/or STV translocation, which is the endpoint for an assay to identify antagonists of HTV and/or STV translocation.
  • the translocation promoting agent produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins may be used as an immunogen to generate antibodies that recognize the translocation promoting agent protein.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • the anti-translocation promoting agent antibodies of the invention may be cross reactive, e.g., they may recognize translocation promoting agent from different species. Polyclonal antibodies have greater likelihood of cross reactivity.
  • an antibody of the invention may be specific for a single form of translocation promoting agent, such as the murine translocation promoting agent.
  • such an antibody is specific for human translocation promoting agent.
  • an antibody of the invention is specific for a masked epitope on the translocation promoting agent that is exposed on binding to HTV or STV.
  • an antibody of the invention is specific for an epitope created by the binding of the translocation promoting agent with HTV or STV, and/or CD4, or both HTV or SIV and CD4.
  • the binding of HTV envelope protein to CD4 induces a conformational change in gpl20 or gpl30, which results in an increased affinity of gpl20 or gpl30 for Bonzo or BOB, and possibly a concomitant unmasking of a Bonzo or BOB epitope.
  • Such antibodies can be selected on the basis of binding under conditions of HIV or STV binding to the translocation promoting agent, e.g., at 4EC to inhibit translocation, and screened for non-binding to the free translocation promoting agent.
  • translocation promoting agent or derivative or analog thereof
  • various host animals can be immunized by injection with the translocation promoting agent, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the translocation promoting agent or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's
  • mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein [Nature 256:495-497 (1975)], as well as the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunology Today 4:72 1983); Cote et al, Proc. Natl. Acad. Sci. U.S.A.
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology described in [PCT US90/02545].
  • techniques developed for the production of "chimeric antibodies" [Morrison et al, J. Bacteriol.
  • Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(abN) 2 fragment which can be produced by pepsin digestion of the antibody molecule; the FabN fragments which can be generated by reducing the disulfide bridges of the F(abN) 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoa
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of a translocation promoting protein, one may assay generated hybridomas for a product which binds to a translocation promoting agent fragment containing such epitope. For selection of an antibody specific to a translocation promoting agent from a particular species of animal, one can select on the basis of positive binding with a translocation promoting agent expressed by or isolated from cells of that species of animal.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the translocation promoting agent, e.g., for Western blotting, imaging translocation promoting agent in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art.
  • Suitable labels for antibodies include enzymes, fluorophores (e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chclated lanthanide series salts, especially Eu 3+ green fluorescent protein, to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and chemiluminescent agents.
  • fluorophores e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chclated lanthanide series salts, especially Eu 3+ green fluorescent protein, to name a few fluorophores
  • chromophores e.g., radioisotopes, chelating agents, dyes, colloidal gold, latex particles,
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 59 Fe, 90 Y, 12 T, 131 I, and 186 Re
  • known currently available counting procedures may be utilized.
  • the label is a protein, e.g., an enzyme or fluorescent protein
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • Direct labels are one example of labels which can be used according to the present invention.
  • a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g., UN. light to promote fluorescence.
  • colored labels include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Patent 4,373,932) and May et al.
  • direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety including proteins comprising such moieties such as green fluorescent protein.
  • indirect labels comprising enzymes can also be used according to the present invention.
  • enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
  • Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.
  • labels for use in the invention include magnetic beads or magnetic resonance imaging labels.
  • a phosphorylation site can be created on an antibody of the invention for labeling with 32 P, e.g., as described in European Patent No. 0372707 (application No. 89311108.8) by Sidney Pestka, or U.S. Patent No. 5,459,240, issued October 17, 1995 to Foxwell et al.
  • proteins, including antibodies can be labeled by metabolic labeling. Metabolic labeling occurs during in vitro incubation of the cells that express the protein in the presence of culture medium supplemented with a metabolic label, such as [ 35 S]-methionine or [ 32 P]-orthophosphate.
  • the invention further contemplates labeling with [ 14 C] -amino acids and [ 3 H] -amino acids (with the tritium substituted at non-labile positions).
  • antibodies that agonize or antagonize the activity of translocation promoting protein can be generated. Such antibodies can be tested using the assays for identifying ligands, for example.
  • Such antibodies when conjugated with a toxin or radioactive element, can be used to target HiN-permissive cells for destruction.
  • cells harboring HTV particularly in its dormant phase, can be destroyed with antibodies, e.g., conjugated to a toxin such as ricin or a radioisotope such as 32 P or I25 I, when such antibodies are specific for the translocation promoting protein.
  • Cell lines expressing CD4 and one or more members of the chemokine receptor family, and more preferably Bonzo or BOB, are infected with an HTV-reporter virus or SrV-reporter virus that is pseudotyped with one or more selected envelope glycoproteins.
  • Compound libraries are assayed for their ability to inhibit infection of the cells by the pseudotyped virus.
  • Candidate compounds are selected and then counter-screened for non-specific effects on infection with virus pseudotyped with non-HTV or non-STV envelope proteins such as MLV amphotropic env or with VSV-G env.
  • Cell lines include, but are not limited to murine 3T3 cells, human HeLa, U87MG, HOS, and 293 cells that are transgenically manipulated to express CD4. Additional human cell lines that do not normally express either CXCR4 or CKR-5 (such as SCL) can also be used. Additional cell lines are listed in Example 1, below.
  • HTV and SIV vectors include, but are not limited to HIV or SIV-luciferase, HTV or SrV-alkaline phosphatase, HIV or SIV- CD24 and HIV or STV-2 LTR-Green Fluorescent Protein.
  • the env gene can be inactivated by frame shifting, and the reporter gene is then inserted to replace the Nef open reading frame. Additional vectors can be made for easier screening in murine cells, in which expression of HTV-LTR-driven reporters is only about 1% of the level in human cells.
  • Such vectors are based on the HIN-gpt prototype (Page et al. 1990), such that the reporter, e.g. luciferase is placed under control of the SV40 promoter within the env gene, ensuring high level expression following integration.
  • HTV-l envelope glycoproteins whose tropism for CCRs and CXR4 have been determined are appropriate for screening for alternative translocating promoting agents.
  • CCR5-tropic envelope glycoproteins ADA and YU-2 (which can also use CCR3) 92US715, and 91US005 and one dual tropic for CXCR4 and CXCR5 (92HT593) are also able to infect cells tranfected with BOB.
  • Two of the envelope proteins for primary HIV-1 strains (the dual tropic 92HT539 and CR5-tropic 92BRO25) also use Bonzo.
  • T-tropic envelope glycoproteins include HXB2, 92UGO21, and 92HT599 also use BOB or Bonzo.
  • CCR5-tropic envelope glycoproteins that do not use either BOB or Bonzo include BaL, 92UG975, 93HT966, 92RWO20, 93TH976, 92BRO20, and 93MW965.
  • JRFL is a CCR5-tropic envelope glycoprotein that uses BOB only weekly.
  • Envelope glycoproteins of STV such as MaclAl 1-Agm TYOl, or Mac 239 can also be used to screen for receptors that are used by STV.
  • translocation promoting agent of the invention provides for expression of translocation promoting agent in quantities greater than can be isolated from natural sources, or in indicator cells that are specially
  • the present invention contemplates an alternative method for identifying agonists and antagonists of HTV translocation directed to modulating the activity of the translocation promoting agent using various screening assays known in the art.
  • such agonists or antagonists competitively inhibit HIN binding, or more particularly, an HIV or STV envelope protein binding to the translocation promoting agent; in another embodiment, the agonist or antagonist indirectly affects HIN translocation, whether by non-competitive binding to the translocation promoting agent, or by affecting the level of expression of the translocation promoting agent.
  • Any screening technique known in the art can be used to screen for antagonists of CD4-HIV or SIV envelope-translocation promoting agent association.
  • the present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and antagonize such activity in vivo.
  • natural products libraries can be screened using assays of the invention for molecules that antagonize HIV-translocation promoting activity.
  • Identification and screening of antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.
  • synthetic libraries [Needels et al., Proc. Natl. Acad. Sci. USA 90:10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993); Lam et al., International Patent Publication No. WO 92/00252; Kocis et al., International Patent Publication No. WO 9428028, each of which is incorporated herein by reference in its entirety], and the like can be used to screen for translocation promoting protein ligands according to the present invention.
  • assays for binding of natural ligand to cells that express recombinant forms of a translocation promoting protein ligand binding domain for Bonzo or BOB for example, can be performed.
  • the soluble ligands can be provided readily as recombinant or synthetic proteins.
  • the screening can be performed with recombinant cells that express the translocation promoting agent, or alternatively, using purified protein, e.g., produced recombinantly, as described above.
  • purified protein e.g., produced recombinantly
  • the ability of a labeled, soluble or solubilized translocation promoting agent that includes the ligand-binding portion of the molecule, to bind ligand can be used to screen libraries, as described in the references cited above.
  • orphan chemokines, potential chemokines, or potential ligands that are obtained from random phage libraries or chemical libraries, as described herein can be tested by any of the numerous assays well known in the art and exemplified herein.
  • an in situ assay is employed in which the detection of the calcium signaling elicited by the binding of a potential chemokine to a chemokine receptor is indicative of the chemokine having specificity for the chemokine receptor, and therefore is a ligand.
  • the component or components of a therapeutic composition of the invention may be introduced parenterally, transmucosally, e.g., orally, nasally, or rectally, or transdermally.
  • administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • an antagonist to the Bonzo and/or BOB (CD4-gpl20-gp41) complex may be introduced by injection into the blood.
  • the antagonist may be a specific antibody raised against the Bonzo and/or BOB (CD4-gpl20-gp41)complex or a mimic to Bonzo or BOB that competitively competes with Bonzo or BOB respectively, for the (CD4-gpl20-gp41)complex.
  • the antagonist can be an antibody to Bonzo.
  • the therapeutic compound can be delivered in a vesicle, in particular a liposome [see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317- 327; see generally ibid.]. To reduce its systemic side effects, this may be a preferred method for introducing an antagonist to Bonzo or BOB.
  • the therapeutic compound can be delivered in a controlled release system.
  • an antibody as described above may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used [see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al, N. Engl. J. Med. 321:574 (1989)].
  • polymeric materials can be used [see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)].
  • a controlled release system can be placed in proximity of a therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose [see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)].
  • the antagonist can be delivered by intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.
  • the antagonist properly formulated, can be administered by nasal or oral administration.
  • a constant supply of the antagonist can be ensured by providing a therapeutically effective dose (i.e., a dose effective to induce metabolic changes in a subject) at the necessary intervals, e.g., daily, every 12 hours, etc.
  • a therapeutically effective dose i.e., a dose effective to induce metabolic changes in a subject
  • these parameters will depend on the severity of the disease condition being treated, other actions, such as diet modification, that are implemented, the weight, age, and sex of the subject, and other criteria, which can be readily determined according to standard good medical practice by those of skill in the art.
  • a subject in whom administration of the antagonist is an effective therapeutic regiment for AIDS is preferably a human, but can be a primate with a related viral condition.
  • the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any primate.
  • a gene encoding a translocation promoting agent, or antisense or ribozyme specific for translocation promoting agent mRNA is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Ban virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Ban virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • macrophage can be specifically targeted.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci. 2:320- 330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. [J. Clin. Invest. 90:626-630 (1992)], and a defective adeno- associated virus vector [Samulski et al., J. Virol. 61:3096-3101 (1987); Samulski et al, J. Virol. 63:3822-3828 (1989)].
  • HSV1 herpes virus 1
  • the gene or antigene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62:1120; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845.
  • a retroviral vector e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U
  • specific PBMCs are removed from an HIV-positive subject animal (e.g., a human) and the gene encoding Bonzo, for example, is replaced by a modified Bonzo gene that retains its natural activity but cannot translocate HTV-2.
  • the subject animal is depleted of its co ⁇ esponding PBMCs (e.g., if the gene modification was performed in a macrophage or T-cell, then the macrophages or T- cell are depleted) and the modified PBMCs are reintroduced into the subject animal.
  • Such an animal subject should then have macrophages or T-cells, which are capable of responding to the appropriate natural ligand for Bonzo, but which are no longer susceptible to HTV-2 translocation/infection, via Bonzo.
  • one or more additional translocation promoting agent genes (such as CC-CKR5 or CXCR4) are modified in a similar manner to more fully block HIN translocation/infection.
  • the vector can be introduced in vivo by lipofection [Feigner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988); Feigner and Ringold, Science 337:387-388 (1989)].
  • Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra].
  • Targeted peptides e.g., hormones or neurofransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990].
  • the present invention extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of translocation promoting agent at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • antisense or ribozyme nucleic acids may be produced chemically, or may be expressed from an "antigen.”
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule [see Marcus-Sekura, Anal. Biochem. 172:298 (1988)]. In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form.
  • antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into organ cells. Antisense methods have been used to inhibit the expression of many genes in vitro [Marcus-Sekura, 1988, supra; Hambor et al., J. Exp. Med. 168:1237 (1988)].
  • Preferably synthetic antisense nucleotides contain phosphoester analogs, such as phosphorothiolates, or thioesters, rather than natural phosphoester bonds. Such phosphoester bond analogs are more resistant to degradation, increasing the stability, and therefore the efficacy, of the antisense nucleic acids.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it [Cech, J. Am. Med. Assoc. 260:3030 (1988)]. Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead”-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target MRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • the DNA sequences encoding the translocation promoting agent can be used to prepare antisense molecules against and ribozymes that cleave mRNAs for translocation promoting agent, thus inhibiting expression of the gene encoding the translocation promoting agent, which can reduce the level of HTV translocation in macrophages and T cells.
  • the transgenic mice of the present application can be produced as detailed in Killeen et al. (1993) EMBO 12 1547-1553, which is hereby incorporated by reference.
  • the construction of the human CD4 + murine CD4 " mice are described by Killeen et al. (1993) EMBO 12 1547-1553.
  • a Bonzo transgene is constructed using a human Bonzo minigene that includes all of the coding region exons and ⁇ 3 kb of sequence (including the first intron) upstream of the coding sequence.
  • B6/SIL F2 eggs or B6/SIL FI x human CD47murine CD4 " eggs are microinjected with the human Bonzo transgene according to standard procedures described by Hogan et al. (1986). Founders are identified by Southern blotting using a human Bonzo cDNA probe.
  • Solid supports include glass beads, sugar beads (SEPHADEX, SEPHAROSE, Agarose, SEPHACEL etc.) magnetic beads, and dowex-type materials.
  • Biological materials may be passed through cells bound to solid supports by common methods know to any person skilled in the art including but not limited by batchwise, by centrifugation, pressure-membrane filtration (e.g. Amicon or Millipore filtration) and through various types of columns.
  • STV receptors Using an expression cloning strategy to identify STV receptors, isolated genes encoding two members of the seven-transmembrane G-protein coupled receptor family were isolated that are used not only by SIN's, but also by strains of HTV-2 and M-tropic HTV-l. Both receptors are closely related to the chemokine receptor family and are expressed in lymphoid tissues. One of the receptors is also expressed in colon and thus may have a role in viral transmission. Use of these new receptors following experimental infection of non-human primates with SIV strains may provide important insight into viral transmission and mechanisms of STV- and HTV-induced acquired immune deficiency syndrome.
  • 10 Fg of the retroviral plasmid DNA was transfected into BOSC23 cells (2xl0 6 cells in a 10-cm culture dish) by calcium phosphate precipitation.
  • the culture supernatant was collected, cleared of cell debris, and then added with 8 mg/ml polybrene to ten 10-cm dishes, each containing 5xl0 5 3T3.CD4 cells. After a 16 hour incubation at 37°C, the viral supernatant was replaced with DMEM-10% fetal calf serum. This high multiplicity infection resulted in 100% transduction of the 3T3.CD4 cells, as assayed in controls in which pMX.mCD4, (whose product is detectable by FACS analysis) was mixed in various ratios with the cDNA library.
  • HTV-puro a selectable replication-defective virus
  • STV agmTY01 or SrV maclA11 Envs a selectable replication-defective virus
  • HTV-puro pseudotypes were generated by transfecting 293T cells with 10 Fg of pHIV-puro (an HTV construct in which env contains a frameshift mutation and nef has been replaced by the SV40 promoter regulating the puromycin resistance gene) and 10 Fg of Env expressing plasmids.
  • Virus-containing supematants were harvested 48 hours post-transfection, filtered, and applied to the library-transduced 3T3.CD4 cells (10 ml viral supernatant per plate). After two days, the 3T3.CD4 cells were replated in 24-well plates (lxlO 5 cells per well) with medium containing 5 Fg/ml puromycin. Selection medium was changed every 2 to 3 days, and visible colonies typically arose in 6 to 9 days. These colonies were then tested for their ability to be infected with HIV-luc pseudotyped with the HTV-l M-tropic Env JRFL or SIV Envs from macl Al 1 or agmTYOl. Colonies infected by JRFL pseudotyped viruses were also stained with anti-CCR5 antibody.
  • Genomic DNA isolation and PCR cloning To recover retrovirus-transduced cDNAs, 50 ng of genomic DNA isolated from each puromycin resistant clone was subjected to PCR. cDNA segments were amplified by using primers complementary to retroviral vector flanking the inserts (upstream 5'GGTGGACCATTCTCTAGACT and downstream 5'CCCT TTTTCTGGAGACTAAAT). The PCR was set up using the Expand kit from Boehringer Mannheim and run for 35 cycles at 58°C annealing temperature. The resulting PCR fragment was purified and cloned into the pCR3.1 vector (Invitrogen) and sequenced. Amino acid sequence alignments were analyzed using the DNASTAR program.
  • Luciferase reporter viruses were prepared in 293T cells as described [Deng et al., 1996, supra; Landau et al., J. Virol., 65:162-169 (1991)] using pNL-Luc-E ' R " vector and Env expression plasmids.
  • Env expression vectors for HTV-l JRFL, ADA, BaL, HXB2, 92UG021, 92US715, 92BR025, 92HT593, as well as SIV mac239, macl Al 1 and agmTYOl have been described [Landau et al., 1991, supra; Westervelt et al., Proc. Natl. Acad.
  • the fflV-2 ST24.1 and p7312A envelope expression plasmids were generated by PCR subcloning of the proviral DNA template into the expression plasmid pSP272.
  • the HIV-2 UC1 envelope expression plasmids were synthesized by published procedures [Evans et al., Science, 240:1522-1525 (1988)] and UC2 [Barnett et al., J.
  • SUBST1TUTESHEET (RULE26) (Coulter) to normalize virus stocks for infection.
  • pseudotyped viruses 50 ng p24 per infection, 5x10 4 cells per well in 24-well plates.
  • 3T3.CD4 cells expressing the new receptors were prepared by retroviral transduction with pMX.Bonzo and pMX.BOB. After 3 days, cells were harvested and resuspended in 120 FI of luciferase lysis buffer (Promega). The luciferase activity in 20 FI of lysate was assayed in a Wallac Microbeta 1450 Counter, using commercially available reagents (Promega).
  • RNA Polyadenylated RNA was prepared from various human cell lines with the Micro-Fast Track Kit (Invitrogen). Samples (5 Fg of RNA) were electrophoresed through a 1% argarose-formaldehyde gel and transfe ⁇ ed to a GeneScreen nitrocellulose membrane. Multiple tissue Northern blot I and II, purchased from Clontech, contains approximately 2 Fg poly(A) + RNA from each tissue. Integrity of blots was assayed by GAPDH probing. Full length cDNAs of BOB and Bonzo were labeled with 32 P by the Random Primed DNA labeling Kit (Boehringer-Mannheim) and used to probe Northern blots.
  • Lymphocyte purification and RT-PCR Monocytes were purified from buffy coats using 46% percoll gradient. To purify T and B cell subsets, PBMC were stained with phycoerythrin conjugated anti-CD3 or anti-CD 19 antibodies (Becton & Dickinson) and sorted using FACS (Coulter).
  • RNA was isolated using RNAzol reagent (Cinna/Biotecx), treated with Rnase-free Dnase, and 0.5 Fg was used for cDNA synthesis using Superscript II RNAse H reverse transcriptase and random hexamer primers (Gibco-BRL); one-twentieth of this reaction was used as a template for PCR amplification with Taq DNA polymerase.
  • BOB primers used for RT-PCR upstream (from ATG) 5'CATCTGCTCTTTGGTGATG, downstream (550 bp from ATG) 5'GTATGGCTTATCATCAATCAGC, amplifies -600 bp transcript; Bonzo primers were: upstream (from 270 bp downstream of ATG) 5'CAGGCATCCATGAATGGGTGT and downstream (from stop codon)
  • SUBST1TUTESHEET(RULE26) 5'CAAGGCCTATAACTGGAACATG CTG amplifies -750 bp transcript.
  • the PCR reaction was run for 30 cycles at 94°C for 40 seconds, 58°C for 40 seconds, and 72°C for 1 min.
  • control cDNA reactions in which reverse transcriptase was omitted were prepared in parallel. These were uniformly negative.
  • the human astroglial U87.CD4 cell line which does not express CCR5 or CXCR4 and is resistant to infection with pseudotypes displaying T-tropic HXB2 or M-tropic JRFL Envs [Clapham et al., Virology, 181:703-715 (1991)], was readily infectable with STV agmTY01 and, to a lesser degree, with STV maclAn Env-pseudotyped viruses (Fig. lb).
  • CEMxl74 a T cell/B cell hybrid that also lacks CCR5 expression and is commonly used to amplify STV in culture [Stefano et al., J.
  • An expression cloning strategy as outlined in Fig. 2a was employed to isolate the genes encoding such receptors.
  • a human T cell clone cDNA library subcloned in the retroviral vector pMX (obtained from DNAX), was transfected into BOSC23 packaging cells and supernatant from these cells was then used to infect 3T3.CD4 cells.
  • the transduced 3T3.CD4 cells were in turn challenged with a selectable replication-defective virus (HTV-puro) pseudotyped with either the SIN agmTY01 or SIN mac i A ii Envs followed by puromycin selection.
  • HTV-puro selectable replication-defective virus
  • a total of 280 puromycin resistant colonies were obtained with the STV agmTY01 infection and about 170 with the SIV maclA11 infection. These colonies were then tested for their ability to be infected with HIN-luc pseudotyped with the HTV-l M-tropic Env JRFL. Representative results after infection of several candidate colonies with virus bearing STV or HIV Envs are shown in Fig. 2b. Approximately 10% of the SIN agmTY01 -selected and 90% of the SIV maclA11 -selected colonies were infectable with the JRFL Env pseudotypes, and all of these JRFL Env infectable colonies were shown by PCR and antibody staining to express CCR5 (e.g.
  • Genomic D ⁇ As extracted from CCR5-negative colonies were subjected to PCR amplification using the primers flanking inserts in the pMX retroviral vectors. A common 2.1 kb band was detected with SIN agmTY01 -selected colonies, and a common 2.2 kb band with Srv maclA1] -selected colonies.
  • the PCR products were subcloned into expression vector pCR3.1 (Invitrogen), and the D ⁇ A sequences were determined.
  • Bonzo The cD ⁇ A selected with the SIN agmTY01 pseudotypes was found to encode a novel protein of 342 amino acids, Bonzo; whereas its counterpart selected with the SIN maclA ⁇ pseudotypes encodes a protein of 360 amino acids, designated BOB (Brother of Bonzo). Comparison of these sequences with those in genome databases indicates that both molecules are members of the large family of G-protein coupled receptors. BOB is identical to a previously cloned orphan receptor, GPR15 [Heiber et al. Genomics, 32:462-465 (1996)], whereas Bonzo is a novel gene with no identity to sequences in the expressed sequence tag (EST) databases.
  • EST expressed sequence tag
  • Both BOB and Bonzo are related to the chemokine receptor family proteins, however, they share only 20-25% amino acid sequence identity with CCR5 and CXCR4. Sequence alignments of these receptors and of CCR5 and CXCR4 are shown in Fig. 2c. Homologues of the SIN receptors from African green monkey and pigtail macaque were also cloned using specific primers. These were found to be 94-97% identical to the human sequences. To test the viral receptor function of the newly identified molecules, their cDNAs were cloned into the pB ABE-puro expression vector and were transiently transfected into human 293T cells along with human CD4. Cells were separately transfected with vectors encoding CCR5 or CCR1 as controls.
  • Transfected cells were then infected with HIV-luc viruses pseudotyped with Envs from a diverse set of SIV, HIV-2, and primary HTV-l isolates.
  • the three SIV envelopes tested were all able to use Bonzo, BOB, and CCR5.
  • the inability of STV Env- pseudotyped virus to infect CCR1 -transfected control cells also shows that none of the STV Envs tested can use CXCR4, which is expressed endogenously in 293T cells.
  • Most of the HIV-2 Envs tested were also able to use BOB (Fig. 3a).
  • Several HTV-2s that grow on transformed T cell lines can also use CXCR4, thus explaining their high background in 293T cells (e.g.
  • Fig. 4a Northern blot hybridization
  • Bonzo cDNA hybridized to a 2.1 kb mRNA species expressed at high levels in spleen, thymus, small intestine, and, to a lesser extent, in peripheral blood leukocytes, prostate and colon. In the placenta, a 2.6 kb species was detected. This species either represents a different homologous gene product, a product of differential splicing, or an antisense mRNA.
  • BOB mRNA was detected in spleen and peripheral blood leukocytes, and weaker expression was detected in thymus and small intestine.
  • BOB mRNA was present in colon, where its high level suggests that it may be expressed in non-lymphoid cells. It will therefore be important to determine if this expression pattern is relevant for sexual transmission of HTV.
  • Bonzo blocked infection of U87.CD4 and CEM cells with HTV-luc pseudotyped with SIN Envs.
  • the expression pattern of Bonzo thus explains the ability of these cells to be infected with African green monkey strains of SIN.

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Abstract

La présente invention se rapporte à de nouveaux agents stimulant la translocation du VIH/VIS (virus de l'immunodéficience humaine/virus de l'immunodéficience simienne), Bonzo et BOB; à des séquences d'ADN et d'acides aminés de la protéine réceptrice Bonzo, ces séquences étant tirées d'un être humain, d'un singe vert d'Afrique et d'un macaque à queue de cochon; à des cellules de mammifères transfectées par Bonzo et/ou BOB, des cellules humaines CD4, ainsi qu'à des anticorps dirigés contre le récepteur Bonzo; à un procédé d'identification d'autres agents stimulants de translocation; enfin, à des utilisations diagnostiques et thérapeutiques de ces agents.
PCT/US1998/014857 1997-07-17 1998-07-17 Recepteurs de structure variable associes a une incursion retrovirale dans des cellules WO1999003888A1 (fr)

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US89615597A 1997-07-17 1997-07-17
US08/896,155 1997-07-17

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001037872A1 (fr) * 1999-11-24 2001-05-31 Millennium Pharmaceuticals, Inc. Anticorps et ligands du recepteur de chimiokine 'bonzo'
WO2001064752A3 (fr) * 2000-03-02 2002-04-18 Univ New York Procedes d'utilisation d'un element facilitant l'entree retrovirale dans les cellules
WO2003037251A3 (fr) * 2001-10-29 2004-03-04 Univ Utah Res Found Activation des bob/gpr15 induite par hivgp120
US7208152B2 (en) 1999-11-24 2007-04-24 Millennium Pharmaceuticals, Inc. Antibodies for “Bonzo” chemokine receptor and therapeutic uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DENG H.-K., ET AL.: "EXPRESSION CLONING OF NEW RECEPTORS USED BY SIMIAN AND HUMAN IMMUNODEFICIENCY VIRUSES.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 388., 17 July 1997 (1997-07-17), United Kingdom, pages 296 - 300., XP002913792, ISSN: 0028-0836, DOI: 10.1038/40894 *
HEIBER M., ET AL.: "A NOVEL HUMAN GENE ENCODING A G-PROTEIN-COUPLED RECEPTOR (GPR15) IS LOCATED ON CHROMOSOME 3.", GENOMICS, ACADEMIC PRESS, SAN DIEGO., US, vol. 32., no. 03., 1 January 1996 (1996-01-01), US, pages 462 - 465., XP002913793, ISSN: 0888-7543, DOI: 10.1006/geno.1996.0143 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001037872A1 (fr) * 1999-11-24 2001-05-31 Millennium Pharmaceuticals, Inc. Anticorps et ligands du recepteur de chimiokine 'bonzo'
US6319675B1 (en) 1999-11-24 2001-11-20 Millennium Pharmaceuticals, Inc. Methods for detecting and/or identifying agents which bind and/or modulate function of “bonzo” chemokine receptor
US7038018B2 (en) 1999-11-24 2006-05-02 Millennium Pharmaceuticals, Inc. Antibodies and ligands for “Bonzo” chemokine receptor
US7208152B2 (en) 1999-11-24 2007-04-24 Millennium Pharmaceuticals, Inc. Antibodies for “Bonzo” chemokine receptor and therapeutic uses thereof
WO2001064752A3 (fr) * 2000-03-02 2002-04-18 Univ New York Procedes d'utilisation d'un element facilitant l'entree retrovirale dans les cellules
WO2003037251A3 (fr) * 2001-10-29 2004-03-04 Univ Utah Res Found Activation des bob/gpr15 induite par hivgp120

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