Method for designing a drug regime for hiv-infected patients

Abstract

The instant disclosure describes a novel genotype and phenotype assay to elucidate and/or evaluate new potential HIV integrase inhibitors, but also currently approved and experimental compounds that target protease, reverse transcriptase, and RNaseH. This assay allows studying linked mutations and mutational patterns that occur under HAART and experimental therapies.

1 . (canceled) 
     
     
         2 . A primer having a nucleotide sequence consisting of any one of SEQ ID NOs: 1-10, 53, and 54. 
     
     
         3 . The primer of  claim 2 , wherein said primer consists of the polynucleotide sequence of any one of SEQ ID NOs: 1-10, 53, and 54. 
     
     
         4 . A set of primers for producing a DNA copy of a segment of an HIV genome comprising the primers having the polynucleotide sequence of any one of SEQ ID NOs: 1-10, 53, and 54 
     
     
         5 . The set of primers of  claim 4  comprising primers having the polynucleotide sequence of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. 
     
     
         6 . The set of primers of  claim 4  comprising primers having the polynucleotide sequence of SEQ ID NO: 53, SEQ ID NO: 2, SEQ ID NO: 54, and SEQ ID NO: 4. 
     
     
         7 . The set of primers of  claim 4  comprising primers having the polynucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 4. 
     
     
         8 . The set of primers of  claim 4  comprising primers having the polynucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 1, and SEQ ID NO: 10. 
     
     
         9 . An in vitro method of obtaining a DNA copy of a segment of an HIV genome comprising:
 a. Obtaining an RNA copy of an HIV genome from a subject infected with HIV;   b. Reverse-transcribing the RNA with any one of the following primer pairs:
 i. SEQ ID NO: 1 and SEQ ID NO: 2, 
 ii. SEQ ID NO: 53 and SEQ ID NO: 2, 
 iii. SEQ ID NO: 5 and SEQ ID NO: 6, or 
 iv. SEQ ID NO: 8 and SEQ ID NO: 9; and 
   c. Amplifying the resulting reverse transcript of:
 i. Subpart b(i) with primers SEQ ID NO: 3 and SEQ ID NO: 4, 
 ii. Subpart b(ii) with primers SEQ ID NO: 54 and SEQ ID NO: 4, 
 iii. Subpart b(iii) with primers SEQ ID NO: 7 and SEQ ID NO: 4, or 
 iv. Subpart b(iv) with primers SEQ ID NO: 1 and SEQ ID NO: 10; and 
   d. obtaining a DNA copy of a segment of an HIV genome.   
     
     
         10 . An in vitro method for designing a drug regimen for an HIV-infected patient by determining the phenotypic susceptibility of HIV to at least one drug, comprising:
 i) using at least one sample comprising HIV RNA from a patient, wherein the sample comprises the complete HIV 5′ LTR-vif coding region;   ii) reverse-transcribing and amplifying the HIV RNA with primers specific for the complete HIV 5′ LTR-vif coding region to obtain at least one amplicon comprising the complete HIV 5′ LTR-vif coding region, wherein at least one primer is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4;   iii) generating a plasmid comprising a reference HIV sequence with a deletion of the complete HIV 5′ LTR-vif coding region;   iv) preparing at least one recombinant virus by recombination or ligation between at least one amplicon obtained in step ii) and the plasmid comprising the reference HIV sequence with a deletion of the complete HIV 5′ LTR-vif coding region obtained in step iii); and   v) monitoring at least one recombinant virus in the presence of at least one drug to determine the phenotypic susceptibility of HIV to at least one drug;   
       wherein said susceptibility is determined by the cytopathogenicity of said recombinant virus to cells or by determining the replicative capacity of said recombinant virus in the presence of at least one drug. 
     
     
         11 . An in vitro method for designing a drug regimen for an HIV-infected patient by determining the phenotypic susceptibility of HIV to at least one drug, comprising:
 i) using at least one sample comprising HIV RNA from a patient, wherein the sample comprises the complete HIV Pol coding region;   ii) reverse-transcribing and amplifying the HIV RNA with primers specific for the complete HIV Pol coding region to obtain at least one amplicon comprising the complete HIV Pol coding region, wherein at least one primer is selected from SEQ ID NO: 53, SEQ ID NO: 2, SEQ ID NO: 54, and SEQ ID NO: 4;   iii) generating a plasmid comprising a reference HIV sequence with a deletion of the complete HIV Pol coding region;   iv) preparing at least one recombinant virus by recombination or ligation between at least one amplicon obtained in step ii) and the plasmid comprising the reference HIV sequence with a deletion of the complete HIV Pol coding region obtained in step iii); and   v) monitoring at least one recombinant virus in the presence of at least one drug to determine the phenotypic susceptibility of HIV to at least one drug;   
       wherein said susceptibility is determined by the cytopathogenicity of said recombinant virus to cells or by determining the replicative capacity of said recombinant virus in the presence of at least one drug. 
     
     
         12 . An in vitro method for designing a drug regimen for an HIV-infected patient by determining the phenotypic susceptibility of HIV to at least one drug, comprising:
 i) using at least one sample comprising HIV RNA from a patient, wherein the sample comprises the complete HIV RT-INT coding region;   ii) reverse-transcribing and amplifying the HIV RNA with primers specific for the complete HIV RT-INT coding region to obtain at least one amplicon comprising the complete HIV RT-INT coding region, wherein at least one primer is selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 4;   iii) generating a plasmid comprising a reference HIV sequence with a deletion of the complete HIV RT-INT coding region;   iv) preparing at least one recombinant virus by recombination or ligation between at least one amplicon obtained in step ii) and the plasmid comprising the reference HIV sequence with a deletion of the complete HIV RT-INT coding region obtained in step iii); and   v) monitoring at least one recombinant virus in the presence of at least one drug to determine the phenotypic susceptibility of HIV to at least one drug;   
       wherein said susceptibility is determined by the cytopathogenicity of said recombinant virus to cells or by determining the replicative capacity of said recombinant virus in the presence of at least one drug. 
     
     
         13 . An in vitro method for designing a drug regimen for an HIV-infected patient by determining the phenotypic susceptibility of HIV to at least one drug, comprising:
 i) using at least one sample comprising HIV RNA from a patient, wherein the sample comprises the complete HIV GAG-PR coding region;   ii) reverse-transcribing and amplifying the HIV RNA with primers specific for the complete HIV GAG-PR coding region to obtain at least one amplicon comprising the complete HIV GAG-PR coding region, wherein at least one primer is selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 1, and SEQ ID NO: 10;   iii) generating a plasmid comprising a reference HIV sequence with a deletion of the complete HIV GAG-PR coding region;   iv) preparing at least one recombinant virus by recombination or ligation between at least one amplicon obtained in step ii) and the plasmid comprising the reference HIV sequence with a deletion of the complete HIV GAG-PR coding region obtained in step iii); and   v) monitoring at least one recombinant virus in the presence of at least one drug to determine the phenotypic susceptibility of HIV to at least one drug;   
       wherein said susceptibility is determined by the cytopathogenicity of said recombinant virus to cells or by determining the replicative capacity of said recombinant virus in the presence of at least one drug. 
     
     
         14 . A primer having a nucleotide sequence consisting of any one of SEQ ID NOs: 20, 24, 25, 36, 38, 40, 42, 44, 55, 56, 57, or 58. 
     
     
         15 . The primer of  claim 14 , wherein said primer is suitable for sequencing a segment of a naturally occurring or recombinant human immunodeficiency virus genome.