Heteroduplex tracking assay
A change in viral tropism occurs in many HIV positive individuals over time and may be indicated by a shift in coreceptor use from CCR5 to CXCR4. The shift in coreceptor use to CXCR4 has been shown to correlate with increased disease progression. In patients undergoing HAART, the predominant populations of virus may be shifted back to CCR5-mediated entry soon after the CXCR4-specific strains have emerged. The present invention relates to a diagnostic method to monitor coreceptor use in the treatment and clinical management of human immunodeficiency virus (HIV) infection. The present invention further relates to a diagnostic method applied to HIV-positive individuals undergoing HAART to monitor the suppression of CCR5- or CXCR4-specific strains. The diagnostic methods may be used to assist in selecting antiretroviral therapy and to improve predictions of disease prognosis over time. The methods of the invention include cell-based methods, including cell fusion assays, and molecular-based methods, including heteroduplex tracking assay, to both quantitatively and qualitatively analyze patient-derived HIV for coreceptor usage.
1. A method of determining whether to initiate antiretroviral therapy in a patient comprising determining the viral load of a population of human immunodeficiency virus (HIV) using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of:
(a) screening the patient-derived biological sample to determine the CCR5 coreceptor usage and the CXCR4 coreceptor usage of individual HIV within the population of HIV;
(b) determining the proportion of HIV using the CCR5 coreceptor (R5) versus the CXCR4 coreceptor (R4) wherein the proportion is expressed as a variable called the Quantity of X4 and R5 (QXR), which represents the fraction of virus in a specimen using the R5 coreceptor;
(c) determining coreceptor specific viral loads of the population of HIV wherein the R5-specific viral load=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR);
wherein an X4-specific viral load is greater than zero indicates a need to initiate antiretroviral therapy in the patient.
2. A method of monitoring the efficacy of antiretroviral therapy in a patient comprising determining the viral load of a population of human immunodeficiency virus (HIV) using the CXCR4 coreceptor (X4-specific viral load) in a patient-derived biological sample comprising the steps of:
(a) screening the patient-derived biological sample to determine the CCR5 coreceptor usage and the CXCR4 coreceptor usage of individual HIV within the population of HIV;
(b) determining the proportion of HIV using the CCR5 coreceptor (R5) versus the CXCR4 coreceptor (R4) wherein the proportion is expressed as a variable called the Quantity of X4 and R5 (QXR), which represents the fraction of virus in a specimen using the R5 coreceptor;
(c) determining coreceptor specific viral loads of the population of HIV wherein the R5-specific viral load=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR)
wherein an increase in X4-specific viral load post antiretroviral therapy indicates disease progression.
3. The method of claim 1 , wherein the patient-derived biological sample is a patient-derived HIV primary isolate.
4. The method of claim 3 , wherein the patient-derived HIV primary isolate comprises individual molecular clones.
5. The method of claim 2 , wherein the patient-derived biological sample is a patient-derived HIV primary isolate.
6. The method of claim 5 , wherein the patient-derived HIV primary isolate comprises individual molecular clones.
7. The method of claim 1 , wherein the screening the patient-derived biological sample is with a heteroduplex tracking assay.
8. The method of claim 2 , wherein the screening the patient-derived biological sample is with a heteroduplex tracking assay.
9. The method of claim 1 , further comprising administering antiretroviral therapy to the patient.
10. The method of claim 1 , wherein the antiretroviral therapy is selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.
11. The method of claim 2 , wherein the antiretroviral therapy is selected from the group consisting of combination antiretroviral therapy (cART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, nonnucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.