Analysis of hiv-1 coreceptor use in the clinical care of hiv-1 infected patients

Abstract

A change in viral tropism occurs in many HIV positive individuals over time and can 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 can be shifted back to CCR5-mediated entry after the CXCR4-specific strains have emerged. The present invention relates to a diagnostic method to monitor coreceptor use in the treatment 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 CXCR4 specific strains. The diagnostic methods can be used to assist in selecting antiretroviral therapy and to improve predictions of disease prognosis over time.

1 - 53 . (canceled)  
     
     
         54 . A diagnostic method of determining CXCR4 and/or CCR5 coreceptor usage in a patient comprising: (a) obtaining patient-derived acquired immunodeficiency virus; and (b) determining usage of the CXCR4 and/or CCR5 coreceptor in said patient-derived acquired immunodeficiency virus, whereby the CXCR4 and/or CCR5 coreceptor usage of the patient is used to determine a suitable antiretroviral treatment regimen.  
     
     
         55 . The diagnostic method according to  claim 54 , wherein the acquired immunodeficiency virus is selected from the group consisting of HIV-1 and HIV-2.  
     
     
         56 . The method according to  claim 54 , where in the patient-derived acquired immunodeficiency virus sample is obtained from peripheral blood.  
     
     
         57 . The diagnostic method according to  claim 54 , wherein the antiretroviral therapy is selected from the group consisting of highly active antiretroviral therapy (HAART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, non-nucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.  
     
     
         58 . The method according to  claim 57 , wherein the nucleoside analogue reverse transcriptase inhibitor is 3TC.  
     
     
         59 . The method according to  claim 57 , wherein the nucleoside analogue reverse transcriptase inhibitor is AZT.  
     
     
         60 . The method according to  claim 57 , wherein the non-nucleoside analogue reverse transcriptase inhibitor is nevirapine.  
     
     
         61 . The diagnostic method according to  claim 54 , wherein said determining step utilizes an indicator cell line to determine said CXCR4 and/or CCR5 coreceptor usage.  
     
     
         62 . The method according to  claim 61 , wherein the indicator cell line comprises an inducible reporter gene construct.  
     
     
         63 . The method according to  claim 62 , wherein the reporter gene construct is selected from the group consisting essentially of green fluorescent protein, placental alkaline phosphatase, firefly luciferase, β-galactosidase and chloramphenicol acetyltransferase.  
     
     
         64 . The method according to  claim 54 , wherein said determining step comprises transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression.  
     
     
         65 . A diagnostic method of determining CXCR4 and/or CCR5 coreceptor usage in a patient comprising: (a) obtaining a clinical specimen from a HIV-infected patient; (b) cloning HIV envelope genes of interest from said clinical specimen; (c) determining usage of said CXCR4 and/or CCR5 coreceptor by said HIV obtained from said clinical specimen using said cloned envelope genes of interest, whereby said CXCR4 and/or CCR5 coreceptor usage by said HIV from said patient specimen is used to determine a suitable antiretroviral treatment regimen.  
     
     
         66 . The diagnostic method according to  claim 65 , wherein the acquired immunodeficiency virus is selected from the group consisting of HIV-1 and HIV-2.  
     
     
         67 . The method according to  claim 65 , where in the patient-derived acquired immunodeficiency virus sample is obtained from peripheral blood.  
     
     
         68 . The diagnostic method according to  claim 65 , wherein the antiretroviral therapy is selected from the group consisting of highly active antiretroviral therapy (HAART), protease inhibitors, fusion inhibitors, integrase inhibitors, coreceptor specific agents, non-nucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors.  
     
     
         69 . The method according to  claim 68 , wherein the nucleoside analogue reverse transcriptase inhibitor is 3TC.  
     
     
         70 . The method according to  claim 68 , wherein the nucleoside analogue reverse transcriptase inhibitor is AZT.  
     
     
         71 . The method according to  claim 68 , wherein the non-nucleoside analogue reverse transcriptase inhibitor is nevirapine.  
     
     
         72 . The diagnostic method according to  claim 65  wherein said determining step utilizes an indicator cell line to determine said CXCR4 and/or CCR5 coreceptor usage.  
     
     
         73 . The method according to  claim 72 , wherein the indicator cell line comprises an inducible reporter gene construct.  
     
     
         74 . The method according to  claim 73 , wherein the reporter gene construct is selected from the group consisting essentially of green fluorescent protein, placental alkaline phosphatase, firefly luciferase, 3-galactosidase and chloramphenicol acetyltransferase.  
     
     
         75 . The method according to  claim 65 , wherein said determining step comprises transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression.  
     
     
         111 - 132 . (canceled)