Polymerase chain reaction assays for monitoring antiviral therapy and making therapeutic decisions in the treatment of acquired immunodeficiency syndrome

Abstract

The present invention relates to methods of monitoring, via polymerase chain reaction, the clinical progression of human immunodeficiency virus infection and its response to antiretroviral therapy. According to the invention, polymerase chain reaction assays may be used to predict immunological decline and to identify, at an early stage, patients whose infection has become resistant to a particular antiretroviral drug regimen.

1 - 4 . (canceled)  
     
     
         5 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) treating the patient with an antiretroviral agent;    (b) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting a plasma sample from the patient;  
 (ii) amplifying HIV-encoding nucleic acid that may be present in the plasma sample using HIV primers via PCR; and  
 (iii) testing for the presence of HIV-encoding nucleic acid sequence in the product of the PCR;  
   (c) correlating positively the presence of HIV-encoding nucleic acid in the product of the PCR with ineffectiveness of the antiretroviral agent; and    (d) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         6 . The method of  claim 5 , wherein the step (c) comprises correlating positively the presence of HIV-encoding nucleic acid in the product of the PCR with an absolute CD4 count of less than about 200 cells per cubic millimeter, which in turn correlates positively with the conclusion that the antiretroviral agent is therapeutically ineffective.  
     
     
         7 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) treating the patient with an antiretroviral agent;    (b) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting a plasma sample from the patient;  
 (ii) amplifying HIV-encoding nucleic acid that may be present in the plasma sample using HIV primers via PCR; and  
 (iii) measuring HIV RNA copy number using the product of the PCR;  
   (c) correlating positively an HIV RNA copy number of greater than about 500 per 200 μl of plasma with the ineffectiveness of the antiretroviral agent; and    (d) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         8 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) treating the patient with an antiretroviral agent;    (b) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting more than one plasma sample from the patient during the treatment;  
 (ii) amplifying HIV-encoding nucleic acid that may be present in the more than one plasma sample using HIV primers via PCR; and  
 (iii) measuring HIV RNA copy numbers using the products of the PCR;  
   (c) correlating positively an increase in HIV RNA copy numbers during the treatment with the ineffectiveness of the antiretroviral agent; and    (d) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         9 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) treating the patient with an antiretroviral agent;    (b) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting more than one plasma sample from the patient at time intervals sufficient to ascertain the existence of a statistically significant decline in plasma HIV RNA copy numbers;  
 (ii) amplifying HIV-encoding nucleic acid that may be present in the more than one plasma sample using HIV primers via PCR;  
 (iii) measuring HIV RNA copy numbers using the products of the PCR; and  
 (iv) comparing the HIV RNA copy numbers in the more than one plasma sample collected during the treatment;  
   (c) correlating positively the absence of a statistically significant decline in plasma HIV RNA copy numbers with the ineffectiveness of the antiretroviral agent; and    (e) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         10 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) collecting a pre-treatment plasma sample from the patient who is about to be treated with an antiretroviral agent;    (b) treating the patient with an antiretroviral agent;    (c) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting a post-treatment plasma sample after initiation of treatment with the antiretroviral agent and at a time interval sufficient to ascertain the existence of a statistically significant decline in plasma HIV RNA copy numbers;  
 (ii) amplifying HIV-encoding nucleic acid that may be present in the pre treatment and post-treatment plasma samples using HIV primers via PCR;  
 (iii) measuring HIV RNA copy numbers using the products of the PCR; and  
 (iv) comparing the HIV RNA copy numbers in the pre-treatment and post treatment plasma samples;  
   (d) correlating positively a ratio of HIV RNA copy numbers in the pre-treatment and the post-treatment plasma samples of less than about 4 to 1 with the ineffectiveness of the antiretroviral agent; and    (e) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         11 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) treating the patient with an antiretroviral agent;    (b) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting a plasma sample from the patient; and  
 (ii) determining whether the plasma sample comprises nucleic acid encoding HIV reverse transcriptase having a mutation at codon 215 or 74, or codons 215 and 74 using HIV primers via PCR;  
   (c) correlating positively the presence of the mutation with an accelerated immunologic decline of said patient compared to patients who do not have the mutation, wherein the accelerated immunologic decline of said patient correlates positively with the ineffectiveness of the antiretroviral agent; and    (d) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         12 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) treating the patient with an antiretroviral agent;    (b) evaluating the effectiveness of the antiretroviral agent by 
 (i) collecting a peripheral blood mononuclear cell (PBMC) sample from the patient;  
 (ii) determining whether the PBMC sample comprises proviral HIV DNA which comprises a mutation at codon 215 or 74, or codons 215 and 74, of the HIV reverse transcriptase gene using HIV primers via PCR;  
   (c) correlating positively the presence of the mutation with an accelerated immunologic decline of said patient compared to patients who do not have the mutation, wherein the accelerated immunologic decline of said patient correlates positively with the ineffectiveness of the antiretroviral agent; and    (d) modifying the patient's therapeutic regimen in response to said correlation.    
     
     
         13 . The method of any one of  claims 5  to  12 , wherein the PCR is performed for not more than 35 cycles.  
     
     
         14 . The method of  claim 13 , wherein the PCR is performed for about 30 cycles.  
     
     
         15 . The method of any one of  claims 5  to  12 , wherein the HIV primers are SK38 and SK39.  
     
     
         16 . The method of any one of  claims 5  to  12 , wherein one HIV primer is primer NE1 (5′TCATTGACAGTCCAGCT-3′) (SEQ ID NO:2).  
     
     
         17 . The method of any one of  claims 5  to  12 , wherein one HIV primer is primer A (5′-TTCCCATTAGTCCTATT-3′) (SEQ ID NO:1).  
     
     
         18 . The method of any one of  claims 5  to  12 , wherein the step of modification comprises increasing the dosage of the antiretroviral agent, changing to another antiretroviral agent, adding one or more additional antiretroviral agents to the patient's therapeutic regimen, or a combination thereof.  
     
     
         19 . The method of any one of  claims 5  to  12 , further comprising a step of treating the patient prophylactically for opportunistic infections using antifungal, antibiotic, and/or antiparasitic medications.  
     
     
         20 . The method of any one of  claims 5  to  12 , wherein the antiretroviral agent is zidovudine or dideoxyinosine.  
     
     
         21 . A method of treating an HIV-infected patient, comprising the steps of: 
 (a) correlating positively the presence of detectable HIV-encoding nucleic acid with a therapeutically ineffective antiretroviral agent; and    (b) modifying the patient's therapeutic regimen in response to said correlation.