Method for designing a drug regime for HIV-infected patients
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. An in vitro method for designing a drug regime 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 reverse transcriptase-integrase coding sequence;
ii) reverse-transcribing and amplifying the HIV RNA with primers specific for the complete HIV reverse transcriptase-integrase coding sequence to obtain at least one amplicon comprising the complete HIV reverse transcriptase-integrase coding sequence, wherein at least one primer comprises one of SEQ ID NO: 4-7;
iii) generating a plasmid comprising a reference HIV sequence with a deletion of the complete HIV reverse transcriptase-integrase coding sequence, wherein said plasmid comprises SEQ ID NO:51;
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 reverse transcriptase-integrase coding sequence 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.
2. An in vitro method for designing a drug regime 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 DNA, wherein the sample comprises the complete HIV reverse transcriptase-integrase coding sequence;
ii) amplifying the HIV DNA with primers specific for the complete HIV reverse transcriptase-integrase coding sequence to obtain at least one amplicon comprising the complete HIV reverse transcriptase-integrase coding sequence, wherein at least one primer comprises one of SEQ ID NO: 4-7;
iii) generating a plasmid comprising a reference HIV sequence with a deletion of the complete HIV reverse transcriptase-integrase coding sequence, wherein said plasmid comprises SEQ ID NO:51;
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 reverse transcriptase-integrase coding sequence 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.