Method of managing the chemotherapy of patients who are HIV positive based on the phenotypic drug sensitivity of human HIV strains

Abstract

The present invention is drawn to a method of managing HIV chemotherapy of patients who are HIV positive, by transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, which sequence encodes at least one desired target enzyme selected from integrase, protease or reverse transcriptase, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of the pol gene, and a HIV-DNA construct that lacks a sequence encoding the desired target enzyme, culturing the transfected cells so as to create a stock of chimeric viruses, assessing the phenotypic sensitivity of the chimeric viruses to at least one inhibitor of the target enzyme encoded by the pol gene of HIV, and assigning a value thereto, constructing a data set containing the value for chimeric virus sensitivity and the corresponding value for a chimeric wild-type strain of HIV, repeating the sensitivity assessment for at least two further inhibitors and thereby constructing at least three such data sets in total, representing the at least one data sets in two dimensional or three dimensional graphical form such that the difference between the chimeric and wild-type sensitivities in the case of each data set provides a visual measure of the resistance of the chimeric stock to treatment by the inhibitor in question, and selecting the optimum inhibitor(s) on the basis of the graphical representation of the resistances so measured.

1 . A method of managing HIV chemotherapy of patients who are HIV positive, which comprises 
 transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, which sequence encodes at least one desired target enzyme selected from integrase, protease or reverse transcriptase, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of said pol gene, and a HIV-DNA construct that lacks a sequence encoding said desired target enzyme,    culturing said transfected cells so as to create a stock of chimeric viruses,    assessing the phenotypic sensitivity of said chimeric viruses to at least one inhibitor of said target enzyme encoded by the pol gene of HIV, and    assigning a value thereto, constructing a data set comprising said value for chimeric virus sensitivity and the corresponding value for a chimeric wild-type strain of HIV,    repeating the sensitivity assessment for at least two further inhibitors and thereby constructing at least three such data sets in total,    representing said at least one data sets in two dimensional or three dimensional graphical form such that the difference between the chimeric and wild-type sensitivities in the case of each data set provides a visual measure of the resistance of the chimeric stock to treatment by the inhibitor in question, and    selecting the optimum inhibitor(s) on the basis of the graphical representation of the resistances so measured.    
     
     
         2 . A method of managing HIV chemotherapy according to  claim 1 , wherein the data sets are represented on a polygonal or quasi-circular graph comprising: 
 (a) a plurality of normalised axes extending radially from an origin, each axis corresponding to one data set or inhibitor or combination thereof;    (b) the axes being normalised such that the sensitivity values for wild-type HIV for the various inhibitors are equal on each axis, the data points for wild-type HIV being optionally represented and connected to form a regular polygon whose vertices lie on the axes and whose center is defined by the origin;    (c) on each axis a data point representing the sensitivity value of the chimeric HIV stock against the inhibitor corresponding to said axis is plotted, the chimeric data points being optionally connected to form a regular or irregular polygon the shape of which represents the resistance of the chimeric stock to a range of inhibitors.    
     
     
         3 . A method according to  claim 2 , wherein each axis has a logarithmic scale.  
     
     
         4 . A method according to  claim 3 , wherein eccentric data points in the chimeric polygon, if represented, identify inhibitors whose usefulness can be assumed to be of little or no benefit to the patient, while data points lying within, on or close outside the wild-type polygon identify inhibitors whose usefulness can be assumed to be of substantial benefit to the patient.  
     
     
         5 . A method according to  claim 1 , wherein each of said three or more data sets further comprises a value for worst-case measurable resistance for the inhibitor in question, said worst case values being represented on said graphical representations such that the data point for the chimeric stock can be compared both to wild-type and to worst-case HIV, thereby providing an assessment of the relative value of the inhibitor in a particular case.  
     
     
         6 . A method of managing HIV chemotherapy of patients who are HIV positive, which comprises the steps of: 
 (a) periodically assessing the phenotypic sensitivity of a patient's HIV strains according to  claim 1;     (b) maintaining the chemotherapy with the selected inhibitor while the patient's HIV strains remain susceptible to the selected chemotherapy;    (c) selecting a different inhibitor if and when the susceptibility of the original inhibitor decreases.    
     
     
         7 . A method according to  claim 1 , wherein the phenotypic sensitivity of said chimeric viruses to inhibitors of at least two enzymes encoded by the pol gene of HIV is simultaneously assessed.  
     
     
         8 . A method according to  claim 1 , wherein said sequence from the pol gene is isolated from a sample of a biological material obtained from the patient whose phenotypic drug sensitivity is being determined.  
     
     
         9 . A method according to  claim 1 , wherein said biological material is selected from plasma, serum or a cell-free body fluid selected from semen and vaginal fluid.  
     
     
         10 . A method according to  claim 1 , wherein the biological material is whole blood to which an RNA stabiliser has been added.  
     
     
         11 . A method according to  claim 1 , wherein the biological material is tissue material selected from brain tissue or lymph nodal tissue.  
     
     
         12 . A method according to  claim 1 , wherein the cell line susceptible to infection by HIV is a CD4 +  T-cell line.  
     
     
         13 . A method according to  claim 12 , wherein the CD4 +  T-cell line is the MT4 cell line or the HeLa CD4 +  cell line.  
     
     
         14 . A method according to  claim 1 , wherein the product of reverse transcription is amplified using a nested PCR technique.  
     
     
         15 . A method according to  claim 14 , wherein at least one outer primer is selected from the group consisting of SEQ ID: 1 and 2.  
     
     
         16 . A method according to  claim 14 , wherein at least one primer selected from the group consisting of SEQ ID: 3, 4, 5 and 6.  
     
     
         17 . A method according to  claim 1 , wherein the transfection is achieved by electroporation.  
     
     
         18 . A method according to  claim 1 , wherein the transfection is achieved by the use of cationic lipids.  
     
     
         19 . A method according to  claim 1 , wherein the phenotypic drug sensitivity of the chimeric viruses to different RT, protease and integrase inhibitors is assessed in an automated cellular-based assay.  
     
     
         20 . A method according to  claim 1 , wherein the phenotypic drug sensitivity of the chimeric viruses and of the wild HIV strain to one or more RT, protease or integrase inhibitor(s) is expressed as an inhibitory concentration (IC value).  
     
     
         21 . A method according to  claim 1 , wherein RT inhibitors are selected from nucleoside RT inhibitors such as AZT, ddI, ddC, 3TC, d4T, 1592U89, non-nucleoside RT inhibitors such as loviride, nevirapine, delavirdine, ateviridine and tivirapine, protease inhibitors such as saquinavir, indinavir and ritonavir and integrase inhibitors such as caffeic acid phenylethyl ester (CAPE), or those described in GB 2,271,566 or WO 95/08540.  
     
     
         22 . A method according to  claim 1 , wherein the patient is drug naïve.  
     
     
         23 . A method according to  claim 1 , wherein the patient has an HIV drug therapy history.  
     
     
         24 . A method according to  claim 1 , wherein the patient derived pol gene has at least one mutation vis a vis the sequence of the laboratory HIV-1 clone HXB2D.  
     
     
         25 . A method according to  claim 1 , further selecting the optimum inhibitor by identifying resistance-associated mutations.