Claims
- 1. An isolated nucleic acid encoding a translocation promoting agent that is substantially homologous to SEQ ID NO: 1.
- 2. The nucleic acid of claim 1 that is obtained from a primate.
- 3. A nucleic acid primer or probe for the nucleic acid of claim 1.
- 4. An isolated nucleic acid encoding a translocation promoting agent having the amino acid sequence selected from the group consisting of SEQ ID NO: 2, and SEQ ID NO: 2 having a conservative substitution.
- 5. The nucleic acid of claim 4 having the nucleic acid sequence of SEQ ID NO: 1.
- 6. A nucleic acid comprising 18 or more nucleotides that hybridizes to the nucleic acid of claim 4 under standard conditions.
- 7. A DNA construct comprising the isolated nucleic acid of claim 4, wherein the isolated nucleic acid is operatively linked to an expression control sequence.
- 8. A method of using the DNA construct of claim 7 to express the translocation promoting agent comprising introducing the construct into a host cell and expressing the translocation promoting agent in said host cell.
- 9. The method of claim 8 further comprising purifying the translocation promoting agent.
- 10. A unicellular host transformed with the recombinant DNA construct of claim 7.
- 11. The unicellular host of claim 8 which is a primate cell.
- 12. An isolated translocation promoting agent having an amino acid sequence that is substantially homologous to SEQ ID NO: 2.
- 13. The translocation promoting agent of claim 12 having the amino acid sequence selected from the group consisting of SEQ ID NO: 2, and SEQ ID NO: 2 having a conservative substitution.
- 14. An antibody to the translocation promoting agent of claim 12.
- 15. The antibody of claim 14 that is a monoclonal antibody.
- 16. An immortal cell line that produces a monoclonal antibody according to claim 15.
- 17. A mammalian cell that expresses human CD4 and is transfected with a vector encoding human Bonzo.
- 18. The mammalian cell of claim 17 which expresses low levels or no CXCR4 and CCR5 in the absence of transduction with a retroviral vector encoding CXCR4 and CCR5.
- 19. The mammalian cell of claim 17 further comprising a vector encoding a translocation promoting agent selected from the group consisting of CCR5, CXCR4, CCR2b, CCR3, and BOB.
- 20. A mammalian cell that is transfected with a vector encoding human CD4 and a vector encoding a human translocation promoting agent selected from the group consisting of Bonzo and BOB.
- 21. The mammalian cell of claim 20 wherein said cell is attached to a solid support matrix.
- 22. The mammalian cell of claim 20 that is a human cell.
- 23. A transgenic non-human mammal comprising a DNA construct containing a human CD4 gene and a DNA construct containing a human translocation promoting agent selected from the group consisting of Bonzo and BOB.
- 24. The transgenic non-human mammal of claim 23 further comprising a DNA construct containing a human translocation promoting agent selected from the group consisting of CCR5, CXCR4, CCR2b, and CCR3.
- 25. The transgenic non-human mammal of claim 23 which is a mouse.
- 26. 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 comprising:
(a) transfecting a mammalian cell with a viral vector containing a human cDNA with a flanking-viral insert; wherein the mammalian cell expresses human CD4 and a known human translocation promoting agent, but does not express the human translocation promoting agent; and wherein the human cDNA is obtained from a human cDNA library; (b) transfecting the mammalian cell with a first selectable viral vector pseudotyped with the specific viral envelope glycoprotein; wherein the first selectable viral vector encodes a first selectable marker; (c) identifying the mammalian cell that expresses the first selectable marker; (d) transfecting the identified mammalian cell with a second selectable viral vector that is pseudotyped with a particular viral envelope glycoprotein; wherein the second selectable viral vector encodes a second selectable marker; and wherein the known human translocation promoting agent can serve in conjunction with CD4 as a receptor for entry of a virus having the particular viral envelope glycoprotein; (e) selecting an identified mammalian cell that does not express the second selectable marker; (f) extracting DNA from the selected mammalian cell; and (g) amplifying the extracted DNA by PCR using primers for the flanking insert from the viral vectors; wherein a nucleic acid encoding the human translocation promoting agent that can serve in conjunction with CD4 as a receptor for entry of a virus having the specific viral envelope glycoprotein is identified.
- 27. The method of claim 26 wherein the first selectable viral vector is a selectable replication-defective virus.
- 28. The method of claim 27 wherein the first selectable viral vector is an pseudotyped vector.
- 29. The method of claim 26 wherein the second selectable viral vector is a selectable replication-defective virus.
- 30. The method of claim 29 wherein the second selectable viral vector is an HIV-pseudotyped vector.
- 31. The method of claim 26 wherein the viral vector containing the human cDNA is obtained from the supernatant of BOSC23 packaging cells after subcloning the human cDNA library in the retroviral vector pMX and transfecting the human cDNA library into the BOSC23 packaging cells.
- 32. The method of claim 26 wherein the mammalian cell is 3T3.CD4.
- 33. The method of claim 26 wherein the first selectable marker confers antibiotic resistance to the cell.
- 34. The method of claim 33 wherein the antibiotic is puromycin and step (c) is performed by administering puromycin to the cells and identifying cells that are puromycin resistant.
- 35. The method of claim 26 wherein either the first selectable marker or the second selectable marker is green fluorescent protein.
- 36. The method of claim 26 wherein the first selectable marker or the second selectable marker is luciferase.
- 37. The method of claim 26 wherein the known translocation promoting agent is selected from the group consisting of CCR5, CXCR4, CCR2b, CCR3, Bonzo, and BOB.
- 38. The method of claim 37 wherein the known translocation promoting agent is CCR5 and the particular viral envelope glycoprotein is JRFL.
- 39. An assay for selecting for a suspected therapeutic agent for possible use in the treatment of AIDS with the use of the cell of claim 17 which comprises:
(a) administering a potential therapeutic agent to the cell; (b) infecting the cell with a virus pseudotyped with an HIV envelope glycoprotein; (c) measuring the ability of the cell to resist said infection; and (d) selecting the potential therapeutic agent when the measured ability of the cell to resist said infection is statistically greater in the presence of said potential therapeutic agent than in the absence of said potential therapeutic agent; wherein said selected potential therapeutic agent is a suspected therapeutic agent.
- 40. The method of claim 39 wherein the virus contains a marker protein and step (c) is performed by detecting the amount of marker protein expressed in the cell; wherein the measured ability of the cell to resist said infection is inversely proportional to the amount of marker protein detected.
- 41. The method of claim 40 wherein the marker protein is selected from the group consisting of luciferase and green fluorescent protein.
- 42. An assay for selecting for a suspected therapeutic agent for possible use in the treatment of AIDS with the use of the cell of claim 20 which comprises:
(a) administering a potential therapeutic agent to the cell; (b) infecting the cell with a virus pseudotyped with an HIV envelope glycoprotein; (c) measuring the ability of the cell to resist said infection; and (d) selecting the potential therapeutic agent when the measured ability of the cell to resist said infection is statistically greater in the presence of said potential therapeutic agent than in the absence of said potential therapeutic agent; wherein said selected potential therapeutic agent is a suspected therapeutic agent.
- 43. The method of claim 42 wherein the virus contains a marker protein and step (c) is performed by detecting the amount of marker protein expressed in the cell; wherein the measured ability of the cell to resist said infection is inversely proportional to the amount of marker protein detected.
- 44. The method of claim 43 wherein the marker protein is selected from the group consisting of luciferase and green fluorescent protein.
- 45. An assay for selecting a plausible therapeutic agent for possible use in the treatment of AIDS with the use of the transgenic non-human mammal of claim 23, comprising:
(a) administering a suspected therapeutic agent to the transgenic non-human mammal; (b) infecting the transgenic non-human mammal with a virus pseudotyped with an HIV envelope glycoprotein; (c) measuring the ability of the transgenic non-human mammal to resist said infection; and (d) selecting the suspected therapeutic agent when the measured ability of the transgenic mammal to resist said infection is statistically greater in the presence of said suspected therapeutic agent than in the absence of said suspected therapeutic agent; wherein said selected suspected therapeutic agent is a plausible therapeutic agent.
- 46. A method of filtering a biological fluid to remove a virus expressing an HIV envelope glycoprotein that binds with CD4 and Bonzo and/or BOB wherein the biological fluid is passed through the cell of claim 21.
- 47. A method of identifying a ligand for human Bonzo comprising:
(a) contacting a potential ligand with a mammalian cell that expresses human Bonzo and human CD4; wherein the mammalian cell does not express CCR5, CXCR4, CCR2b, CCR3, and BOB; (b) transfecting the mammalian cell with a selectable replication-defective HIV virus pseudotyped with a specific viral envelope glycoprotein; wherein Bonzo in conjunction with CD4, serves as a receptor for the entry into a cell of a virus having the specific viral envelope glycoprotein; and (c) detecting the marker protein; wherein a ligand is selected when the amount of the marker protein detected is less than that detected when step (b) is performed without performing step (a).
- 48. The method of claim 47 wherein the marker protein is selected from the group consisting of luciferase and green fluorescent protein.
- 49. The method of claim 47 further comprising:
(d) contacting the ligand with purified human Bonzo; and (e) detecting the binding of the ligand to purified human Bonzo; wherein a ligand is identified that binds to purified human Bonzo.
- 50. A method of identifying a ligand for human BOB comprising:
(a) contacting a potential ligand with a mammalian cell that expresses human BOB and human CD4; wherein the mammalian cell does not express CCR5, CXCR4, CCR2b, CCR3, and Bonzo; (b) transfecting the mammalian cell with a selectable replication-defective HIV virus pseudotyped with a specific viral envelope glycoprotein; wherein BOB in conjunction with CD4, serves as a receptor for the entry into a cell of a virus having the specific viral envelope glycoprotein; and (c) detecting the marker protein; wherein a ligand is selected when the amount of the marker protein detected is less than that detected when step (b) is performed without performing step (a).
- 51. The method of claim 50 wherein the marker protein is selected from the group consisting of luciferase and green fluorescent protein.
- 52. The method of claim 50 further comprising:
(d) contacting the ligand with purified human BOB; and (e) detecting the binding of the ligand to purified human BOB; wherein a ligand is identified that binds to purified human BOB.
- 53. A method of identifying an agent that can enhance the immune response to a specific pathogen or against a specific vaccine comprising:
(a) contacting an agent with a cell; wherein the cell encodes Bonzo/STRL33; and (b) determining the amount of Bonzo/STRL33 expressed by a cell in the presence of the agent; wherein the agent is identified as an agent that can enhance the immune response to a specific pathogen or against a specific vaccine when the amount of expression of Bonzo/STRL33 increases in the presence of the agent relative to in its absence.
- 54. The method of claim 53 wherein said determining is performed with an antibody raised against Bonzo/STRL33.
- 55. The method of claim 53 wherein said determining is performed by PCR.
- 56. An agent obtained by the method of claim 53.
- 57. A method of enhancing the immune response for a specific pathogen or to enhance the effect of a specific vaccine comprising administering the agent of claim 56 to an animal subject.
- 58. A method of identifying an agent that can enhance the immune response to a specific pathogen or against a specific vaccine comprising:
(a) contacting an agent with a cell; wherein the cell normally encodes Bonzo/STRL33 but the coding sequence for Bonzo/STRL33 has been replaced by a coding sequence for a reporter gene; and (b) determining the amount of reporter gene expressed by a cell in the presence of the agent; wherein the agent is identified as an agent that can enhance the immune response to a specific pathogen or against a specific vaccine when the amount of expression of the reporter gene increases in the presence of the agent relative to in its absence.
- 59. The method of claim 58 wherein the coding sequence for the reporter gene encodes green fluorescent protein.
- 60. The method of claim 58 wherein the coding sequence for the reporter gene encodes luciferase.
- 61. An agent obtained by the method of claim 58.
- 62. A method of enhancing the immune response for a specific pathogen or to enhance the effect of a specific vaccine comprising administering the agent of claim 61 to an animal subject.
- 63. A method of identifying an agent that can inhibit the recruitment of memory cells comprising:
(a) contacting an agent with a cell; wherein the cell encodes Bonzo/STRL33; and (b) determining the amount of Bonzo/STRL33 expressed by a cell in the presence of the agent; wherein the agent is identified as an agent that can inhibit the recruitment of memory cells when the amount of expression of Bonzo/STRL33 decreases in the presence of the agent relative to in its absence.
- 64. The method of claim 63 wherein said determining is performed with an antibody raised against Bonzo/STRL33.
- 65. The method of claim 63 wherein said determining is performed by PCR.
- 66. An agent obtained by the method of claim 63.
- 67. A method of treating inflammation comprising administering the agent of claim 66 to an animal subject in need of such treatment.
- 68. A method of identifying an agent that can inhibit the recruitment of memory cells comprising:
(a) contacting an agent with a cell; wherein the cell normally encodes Bonzo/STRL33 but the coding sequence for Bonzo/STRL33 that has been replaced by a coding sequence for a reporter gene; and (b) determining the amount of reporter gene expressed by a cell in the presence of the agent; wherein the agent is identified as an agent that can inhibit the recruitment of memory cells when the amount of expression of Bonzo/STRL33 decreases in the presence of the agent relative to in its absence.
- 69. The method of claim 68 wherein the coding sequence for the reporter gene encodes green fluorescent protein.
- 70. The method of claim 68 wherein the coding sequence for the reporter gene encodes luciferase.
- 71. An agent obtained by the method of claim 68.
- 72. A method of treating inflammation comprising administering the agent of claim 71 to an animal subject in need of such treatment.
- 73. A method of enhancing the immune response for a specific pathogen or to enhance the effect of a specific vaccine comprising administering to an animal subject an agent that causes an increase in Bonzo/STRL 33 expression and/or function.
- 74. A method of treating inflammation comprising administering to an animal subject in need of such treatment an agent that causes a decrease in Bonzo/STRL 33 expression and/or function.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present Application is a continuation-in-part of copending Ser. No. 09/116,498, filed Jul. 16, 1998 which is a non-provisional application claiming priority to Provisional Patent Application Ser. No. 60/052,827 filed Jul. 17, 1997, the disclosures of which are hereby incorporated by reference in their entireties. Applicants claim the benefits of these applications under 35 U.S.C. §§119 (e) and 120.
GOVERNMENT SUPPORT
[0002] The research leading to the present inventions was funded in part by Grant No. RO1 AI 33303 from the National Institutes of Health. The government may have certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60052827 |
Jul 1997 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
09116498 |
Jul 1998 |
US |
Child |
09852156 |
May 2001 |
US |