Assays And Kits For Determining HIV-1 Tropism

• US 20100240024A1
• Filed: 09/02/2009
• Published: 09/23/2010
• Est. Priority Date: 09/03/2008
• Status: Abandoned Application

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Laffler, Thomas G.
• Application Number:
  US12/552,444
• Publication Number:
  US 20100240024A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G01N 2333/715 for cytokines; for lymphokines; for interferons
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/56988 HIV or HTLV

Abstract

The present invention relates to assays and kits for determining tropism of HIV-1 for a chemokine receptor in a test sample obtained from a subject.

Claims

1. An assay for detecting changes in tropism of an HIV-1 population in a test sample obtained from a subject, the assay comprising the steps of:

(a) contacting the test sample obtained from the subject with a first specific binding partner and a second specific binding partner to form a first specific binding partner-HIV-1 complex and a second specific binding partner-HIV-1 complex, wherein the first specific binding partner comprises a CCR5 co-receptor protein or a CCR5 analog thereof and the second specific binding partner comprises a CXCR4 co-receptor protein or a CXCR4 analog thereof;

(b) separating the first specific binding partner-HIV-1 complex and second specific binding partner HIV-1 complex from the test sample;

(c) isolating RNA from the first specific binding partner-HIV-1 complex and RNA from the second specific binding partner-HIV-1 complex;

(d) quantifying the isolated RNA from the first specific binding partner-HIV-1 complex and the isolated RNA from the second specific binding partner-HIV-1 complex and determining a ratio of HIV-1 virus binding to CCR5 co-receptor protein to HIV-1 virus binding to CXCR4 co-receptor protein; and

(e) comparing the ratio determined in step (d) with a predetermined ratio of HIV-1 virus binding to a CCR5 co-receptor protein to HIV-1 virus binding to a CXCR4 co-receptor protein, wherein a change in the ratio compared to the predetermined ratio indicates a change in tropism in the HIV-1 population in the subject.

2. The assay of claim 1, wherein the isolated RNA from the first specific binding partner-HIV-1 complex and the isolated RNA from the second specific binding partner-HIV-1 complex are quantified using PCR, nucleic acid sequence based amplification, tissue microarrays, transcription mediated amplification, nucleic acid sequence based amplification or Northern Blot analysis.

3. The assay of claim 2, wherein the PCR is selected from the group of reverse transcriptase PCR, competitive RT-PCR, real time RT-PCR or differential display RT-PCR.

4. An assay for monitoring whether a subject infected with an HIV-1 population has become tropic for a CXCR4 co-receptor, the method comprising the steps of:

(a) contacting a test sample obtained from the subject with a first specific binding partner and a second specific binding partner to form a first specific binding partner-HIV complex and a second specific binding partner-HIV-1 complex, wherein the first specific binding partner comprises a CCR5 co-receptor protein or CCR5 analog thereof and the second specific binding partner comprises a CXCR4 co-receptor protein or a CXCR4 analog thereof, further wherein said subject was previously identified as HIV-1 tropic for the CCR5 co-receptor;

(b) separating the first specific binding partner-HIV-1 complex and second specific binding partner HIV-1 complex from the test sample;

(c) isolating RNA from the first specific binding partner-HIV-1 complex and RNA from the second specific binding partner-HIV-1 complex;

(d) quantifying the isolated RNA from the first specific binding partner-HIV-1 complex and the isolated RNA from the second specific binding partner-HIV-1 complex, and determining a ratio of HIV-1 virus binding to CCR5 co-receptor protein to HIV-1 virus binding to CXCR4 co-receptor protein; and

(e) comparing the ratio determined in step (d) with a predetermined ratio of HIV-1 virus binding to a CCR5 co-receptor protein or CCR5 analog thereof to HIV-1 virus binding to a CXCR4 co-receptor protein or CXCR4 analog thereof, wherein an increase in the ratio of CXCR4 co-receptor protein or CXCR4 analog thereof compared to the predetermined ratio indicates a change in tropism of the HIV-1 population from the CCR5 co-receptor protein to the CXCR4 co-receptor protein in the subject.

5. The assay of claim 4, wherein the isolated RNA from the first specific binding partner-HIV-1 complex and the second specific binding partner-HIV-1 complex is quantified using PCR, nucleic acid sequence based amplification, tissue microarrays, transcription mediated amplification or Northern Blot analysis.

6. The assay of claim 5, wherein the PCR is selected from the group of reverse transcriptase PCR, competitive RT-PCR, real time RT-PCR or differential display RT-PCR.

7. The assay of claim 4, wherein the subject has been receiving treatment for the HIV-1 with one or more pharmaceutical compositions.

8. The assay of claim 7, wherein the one or more pharmaceutical compositions is a CCR5 antagonist.

9. An assay for monitoring whether an HIV-1 population has become tropic for a CXCR4 co-receptor, the method comprising the steps of:

(a) contacting a test sample obtained from a subject with a specific binding partner to form a first specific binding partner-HIV-1 complex, wherein the specific binding partner comprises a CXCR4 co-receptor protein or a CXCR4 analog thereof, and further wherein the specific binding partner is immobilized on a solid phase;

(b) separating the specific binding partner-HIV-1 complex from the test sample;

(c) isolating RNA from the specific binding partner-HIV-1 complex;

(d) quantifying the isolated RNA from the specific binding partner-HIV-1 complex and total HIV-1 RNA in the test sample and determining a ratio of RNA virus binding CXCR4 co-receptor protein to total HIV-1 RNA;

(e) comparing the ratio determined in step (d) with a predetermined ratio of HIV-1 virus binding to a CXCR4 co-receptor protein or CXCR4 analog thereof to total HIV-1 RNA, wherein an increase in the ratio of step (d) compared to the predetermined ratio indicates that the HIV-1 population has become tropic for the CXCR4 co-receptor protein.

10. The assay of claim 9, wherein the solid phase is selected from the group consisting of a magnetic particle, a non-magnetic particle, a membrane, a ligand, a receptor, a microparticle and a bead.

11. The assay of claim 9, wherein the isolated RNA from the specific binding partner-HIV-1 complex is quantified using PCR, nucleic acid sequence based amplification, tissue microarrays, transcription mediated amplification or Northern Blot analysis.

12. The assay of claim 11, wherein the PCR is selected from the group of reverse transcriptase PCR, competitive RT-PCR, real time RT-PCR or differential display RT-PCR.

13. The assay of claim 9, wherein the subject has been receiving treatment for the HIV-1 with one or more pharmaceutical compositions.

14. The assay of claim 13, wherein the one or more pharmaceutical compositions is a CCR5 antagonist.

15. An assay for monitoring whether an HIV-1 population has become tropic for a CXCR4 co-receptor, the method comprising the steps of:

(a) contacting a test sample obtained from a subject with a specific binding partner to form a first specific binding partner-HIV complex, wherein the specific binding partner comprises a CXCR4 co-receptor protein or a CXCR4 analog thereof, and further wherein the specific binding partner is immobilized on a solid phase;

(b) separating the specific binding partner-HIV-1 complex from the test sample;

(c) isolating RNA from the specific binding partner-HIV-1 complex;

(d) quantifying the isolated RNA from the specific binding partner-HIV-1 complex; and

(e) comparing the quantity of RNA from the specific binding partner-HIV-1 complex with a predetermined level of RNA binding to CXCR4 co-receptor protein or CXCR4 analog thereof, wherein if the isolated RNA quantified from the specific binding partner-HIV-1 complex is less than the predetermined level, then the HIV-1 population has not become tropic for the CXCR4 co-receptor protein, and further wherein if the isolated RNA quantified from the specific binding partner-HIV-1 complex is greater than the predetermined level, then the HIV-1 population has become tropic for the CXCR4 co-receptor protein.

16. The assay of claim 15, wherein the solid phase is selected from the group consisting of a magnetic particle, a non-magnetic particle, a membrane, a ligand, a receptor, a microparticle and a bead.

17. The assay of claim 15, wherein the isolated RNA from the specific binding partner-HIV-1 complex is quantified using PCR, nucleic acid sequence based amplification, tissue microarrays, transcription mediated amplification or Northern Blot analysis.

18. The assay of claim 17, wherein the PCR is selected from the group of reverse transcriptase PCR, competitive RT-PCR, real time RT-PCR or differential display RT-PCR.

19. The assay of claim 15, wherein the subject has been receiving treatment for the HIV-1 with one or more pharmaceutical compositions.

20. The assay of claim 19, wherein the one or more pharmaceutical compositions is a CCR5 antagonist.

21. A kit for determining tropism of an HIV-1 population in a test sample obtained from a subject, the kit comprising:

(a) a first specific binding partner comprising a CXCR4 co-receptor or a CXCR4 analog thereof;

(b) at least one solid phase; and

(c) instructions for using said kit.

22. The kit of claim 21, further comprising a second specific binding partner comprising a CCR5 co-receptor or a CCR5 analog thereof.

23. A kit for determining tropism of an HIV-1 population in a test sample obtained from a subject, the kit comprising:

(a) a first specific binding partner comprising a CCR5 co-receptor or a CCR5 analog thereof;

(b) at least one solid phase; and

(c) instructions for using said kit.

24. The kit of claim 23, further comprising a second specific binding partner comprising a CXCR4 co-receptor or a CXCR4 analog thereof.