VPR function and activity

• US 6,838,236 B1
• Filed: 12/15/1993
• Issued: 01/04/2005
• Est. Priority Date: 02/19/1993
• Status: Active Grant

• Current Assignee: Pennsylvania University of The Trustees of The, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: The Wistar Institute, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Refaeli, Yosef, Levy, David Nathan, Weiner, David B.
• Primary Examiner(s): Housel, James
• Assistant Examiner(s): PARKIN, JEFFREY S
• Application Number:
  US08/167,608
• Time in Patent Office: 4,038 Days
• Field of Search: 424/185.1, 424/186.1, 424/188.1, 424/208, 435/7, 435/7.9, 435/69.3, 435/974, 435/975, 530/350, 530/826
• US Class Current: 435/5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2740/16322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: G01N 33/56988 HIV or HTLV

Abstract

Pharmaceutical compositions comprising the HIV protein vpr or nucleic acid molecule encoding vpr are disclosed. Also disclosed are methods of treating patients suffering from diseases characterized by hyperproliferating undifferentiated cells such as cancer by administering such compositions. Methods of identifying compounds which have anti-HIV activity are disclosed, in particular, methods of identifying compounds which modulate the activity of vpr and of identifying compounds which inhibit vpr binding to the HIV protein gag.

Claims

1. Isolated human immunodeficiency virus Vpr protein produced in eukaryotic cells.

4. The isolated protein of claim 1 wherein said protein is immobilized to a solid phase support.

5. The isolated protein of claim 4, wherein said solid phase support is selected from the group consisting of:

nitrocellulose paper, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

6. The isolated protein of claim 4, wherein said solid phase support is nitrocellulose paper or polystyrene.

2. A method of identifying whether or not an individual has been infected with human immunodeficiency virus comprising the steps of:

a) contacting a test sample from said individual with isolated human immunodeficiency virus Vpr protein produced in eukaryotic cells, and

b) detecting the presence of anti-Vpr antibodies bound to said Vpr, wherein the presence of anti-Vpr antibodies indicates that said individual has been infected with human immunodeficiency virus and the absence of said anti-Vpr antibodies indicates that said individual has not been infected with human immunodeficiency virus.

7. The method of claim 2 wherein said test sample is selected from the group consisting of:

blood, cerebral spinal fluid, amniotic fluid, lymph, semen or vaginal fluid.

8. The method of claim 2 wherein said test sample is blood.

9. The method of claim 2 wherein said Vpr protein is immobilized to a solid phase support.

10. The method of claim 9 wherein said solid phase support is selected from the group consisting of:

nitrocellulose paper, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

11. The method of claim 9 wherein said solid phase support is nitrocellulose paper or polystyrene.

12. The method of claim 2 comprising the steps of:

a) contacting said test sample with Vpr protein that is immobilized to a solid phase support, and

b) the presence of anti-Vpr antibodies bound to said Vpr is detected by

i) adding anti-human antibody antibodies to said test sample and Vpr protein that is immobilized to a solid phase support; and

ii) detecting said anti-human antibody antibodies bound to anti-Vpr antibodies that are bound to Vpr protein that is immobilized said solid phase support.

13. The method of claim 12 wherein said anti-human antibodies are labelled.

14. The method of claim 2 further comprising the step of performing a positive control assay, said positive control assay comprising the steps of:

a) contacting a positive control sample with Vpr protein produced in eukaryotic cells, wherein said positive control sample comprises positive control antibodies which are antibodies that bind to Vpr produced in eukaryotic cells; and

b) detecting the presence of said positive control antibodies bound to said Vpr produced in eukaryotic cells.

15. The method of claim 2 comprising the steps of:

a) contacting said test sample with Vpr protein that is immobilized to a solid phase support, and

b) detecting the presence of anti-Vpr antibodies bound to said Vpr by;

i) washing said solid phase support;

ii) adding to said test sample and Vpr protein that is immobilized to said solid phase support, labelled antibodies that bind to human antibody Fc region;

iii) washing said solid phase support;

iv) detecting said anti-human antibodies bound to said solid phase support.

16. The method of claim 15 wherein said anti-human antibodies are horse radish peroxidase labelled goat anti-human antibodies.

3. A kit for identifying whether or not an individual has been infected with human immunodeficiency virus comprising

a) a first container comprising isolated human immunodeficiency virus Vpr protein produced in eukaryotic cells, and

b) a second container which contains antibodies which specifically bind to Vpr protein produced in eukaryotic cells.

17. The kit of claim 3 wherein said Vpr protein is immobilized to a solid phase support.

18. The kit of claim 17 wherein said solid phase support is selected from the group consisting of:

nitrocellulose paper, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

19. The kit of claim 17 wherein said solid phase support is nitrocellulose paper or polystyrene.

20. The kit of claim 3 further comprising a positive control which contains antibodies which specifically bind Vpr produced in eukaryotic cells.

21. An in vitro method of identifying individuals who have been exposed to the human immunodeficiency virus comprising the following steps:

(i) preparing a test sample from said individual;

(ii) admixing the test sample of step (i) with isolated eukaryotically expressed human immunodeficiency virus Vpr;

(iii) detecting the presence of anti-Vpr antibodies bound to said Vpr;

wherein the presence of said anti-Vpr antibodies indicates that said individual has been exposed to the human immunodeficiency virus.

22. The method of claim 21 wherein said test sample is selected from the group consisting of:

blood, cerebral spinal fluid, amniotic fluid, lymph, semen or vaginal fluid.

23. The method of claim 21 wherein said test sample is blood.

24. The method of claim 21 wherein said Vpr protein is immobilized to a solid phase support.

25. The method of claim 24 wherein said solid phase support is selected from the group consisting of:

nitrocellulose paper, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

26. The method of claim 24 wherein said solid phase support is nitrocellulose paper or polystyrene.

27. An in vitro method of identifying individuals who have been exposed to the human immunodeficiency virus comprising the following steps:

(i) preparing a test sample from said individual;

(ii) admixing the test sample of step (i) with isolated eukaryotically expressed human immunodeficiency virus Vpr immobilized to a solid phase support;

(iii) allowing antibodies, if present in the test sample, to bind to and form an immune complex with said immobilized Vpr;

(iv) detecting the presence of anti-Vpr antibodies bound to said Vpr by admixing a labeled anti-human secondary antibody with the immune complexes of step (iii);

wherein the presence of said anti-Vpr antibodies indicates that said individual has been exposed to the human immunodeficiency virus.

28. The method of claim 27 wherein said anti-human secondary antibody is a goat anti-human antibody labeled with horseradish peroxidase.

29. A diagnostic kit for identifying individuals who have been exposed to the human immunodeficiency virus comprising the following components:

(i) a container comprising isolated eukaryotically expressed human immunodeficiency virus Vpr

(ii) a container comprising a labeled anti-human secondary antibody;

(iii) a container comprising Vpr-specific antibodies;

(iv) a container comprising antibodies that do not bind to Vpr; and

,

(v) a container comprising detection reagents.

30. The kit of claim 29 wherein said Vpr is immobilized to a solid-phase support.

31. The kit of claim 30 wherein said solid phase support is selected from the group consisting of:

nitrocellulose paper, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamide, agarose, and magnetite.

32. The kit of claim 30 wherein said solid phase support is nitrocellulose paper or polystyrene.