Methods of monitoring treatment effectiveness in HIV-infected patients receiveing intensified HAART regimens by measuring episomal 2-LTR circles

1. A method of detecting the presence of replication competent human immunodeficiency virus 1 (HIV-1) virus in a subject who is being treated with an intensified highly active anti-retroviral therapy (HAART) regimen, the method comprising:

selecting a subject who is being treated with an intensified HAART regimen including an integration inhibitor;

obtaining a sample comprising blood;

specifically amplifying a segment spanning two-long terminal repeat (2-LTR) junction of 2-LTR circles using polymerase chain reaction (PCR) to determine a level of 2-LTR circles in the sample; and

determining the presence of replication competent HIV-1 virus based on the level of 2-LTR circles in the sample.

2. The method of claim 1 , further comprising determining that the subject has no detectable plasma levels of viral RNA.

3. The method of claim 2 , comprising determining that the subject has fewer than about 50 copies/ml plasma of viral RNA.

4. The method of claim 1 , wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

5. The method of claim 1 , wherein the integration inhibitor is raltegravir or elvitegravir.

6. The method of claim 1 , wherein the intensified HAART regimen also includes a protease inhibitor.

7. A method of monitoring an intensified HAART regimen in a subject infected with HIV-1, the method comprising:

selecting a subject who is being treated with an intensified HAART regimen including an integration inhibitor;

obtaining a first sample comprising blood from the subject;

determining a first level of 2-LTR circles in the first sample by specifically amplifying a segment spanning 2-LTR junction of 2-LTR circles using PCR;

obtaining a second sample comprising blood from the same subject at least one month after obtaining the first sample;

determining a second level of 2-LTR circles in the second sample by specifically amplifying a segment spanning 2-LTR junction of 2-LTR circles using PCR; and

comparing the second level with the first level of 2-LTR circles,

determining that the intensified HAART regimen is effective when there is a decrease in the second level of 2-LTR circles, or ineffective when there is no change or an increase in the second level of 2-LTR circles, when compared with the first level of 2-LTR circles.

8. The method of claim 7 , wherein the second sample is obtained at least six months after obtaining the first sample.

9. The method of claim 7 , wherein the integration inhibitor is raltegravir or elvitegravir.

10. The method of claim 7 , wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

11. The method of claim 7 , further comprising determining that the subject has no detectable plasma levels of viral RNA.

12. The method of claim 9 , comprising determining that the subject has fewer than 50 copies/ml plasma of viral RNA.

13. A method of determining the effectiveness of an intensification agent for a subject who is being treated with standard HAART for HIV-1 infection, the method comprising:

selecting a subject who is being treated with standard HAART;

obtaining a first sample comprising blood from the subject;

determining a baseline level of 2-LTR circles in the first sample by specifically amplifying a segment spanning 2-LTR junction of 2-LTR circles using PCR;

adding an intensification agent comprising an integration inhibitor to the standard HAART treatment;

obtaining a second sample comprising blood from the subject at two weeks after addition of the intensification agent;

determining a treatment level of 2-LTR circles in the second sample by specifically amplifying a segment spanning 2-LTR junction of 2-LTR circles using PCR;

comparing the treatment level with the baseline level of 2-LTR circles; and

determining that the intensification agent is effective when the treatment level of 2-LTR circles determined at two weeks after addition of the intensification agent is greater than the baseline level.

14. The method of claim 13 , wherein the integration inhibitor is raltegravir or elvitegravir.

15. The method of claim 13 , wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

16. The method of claim 13 , further comprising determining that the subject has no detectable plasma levels of viral RNA.

17. A method of determining the effectiveness of an intensification agent for a subject who is being treated with standard HAART for HIV-1 infection, the method comprising:

selecting a subject who is being treated with standard HAART;

obtaining a first sample comprising blood from the subject;

determining a baseline level of 2-LTR circles in the first sample by specifically amplifying a segment spanning 2-LTR junction of 2-LTR circles using PCR;

adding an intensification agent comprising an integration inhibitor to the standard HAART treatment;

obtaining a second sample comprising blood from the subject at six months after addition of the intensification agent;

determining a treatment level of 2-LTR circles in the second sample by specifically amplifying a segment spanning 2-LTR junction of 2-LTR circles using PCR;

comparing the treatment level with the baseline level of 2-LTR circles; and

determining that the intensification agent is effective when the treatment level of 2-LTR circles determined at six months after addition of the intensification agent is lower than the baseline level.

18. The method of claim 17 , wherein the integration inhibitor is raltegravir or elvitegravir.

19. The method of claim 17 , wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

20. The method of claim 17 , further comprising determining that the subject has no detectable plasma levels of viral RNA.