Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients

Rapid Enzymatic Test for Phenotypic HIV Protease Drug Resistance

“…In a first approach, patient-derived PR has been expressed in E. coli using a similar procedure, starting from patient DNA and measuring the recombinant HIV protease activity by HPLC analysis of the cleavage products of a peptide substrate (Gehringer et al, 2003). It was shown that the concentration of the respective PI resulting in a 50% enzyme inhibition (IC 50 ) correlates well with clinical parameters and blind genotypic analysis.…”

“…Taking advantage of the high acid stability of PR, the enzyme was purified in a one-step acid procedure (von der Helm et al, 1994). As reference for the establishment of the test system, recombinant HIV-1 wild-type PR (clade B MVP899-87 isolate; Gürtler et al, 1994) was expressed and purified as above and then further purified in a second step resulting in > 70% purity (von der Helm et al, 1994;Gehringer et al, 2003). Preparation of recombinant PR from patient plasma takes 5 days and can be paused and continued later at several preparation levels of the protocol.…”

“…To evaluate the potential of our test system for resistance screening, we investigated the inhibition of recombinantly expressed HIV PR from 8 patients that had been geno- typically classified and scored by nucleotide sequencing (Gehringer et al, 2003). The respective resistance of patient-derived PR to PI in our assay was expressed by enzymatic resistance factors (R enz ) reflecting the ratio K i (patient)/K i (WT).…”

“…It has been previously shown by the HPLC-based activity assay that in mixtures of resistant PR and WT PR the ratio between different 'quasispecies' remained unchanged by simultaneous expression of their genes in E. coli (Gehringer et al, 2003). We determined the resistance factors with our fluorogenic assay after mixing WT PR with different proportions of highly resistant patientderived PR.…”

“…In these clinical situations, a sensitive detection of viral subpopulations within a short diagnostic time seems highly desirable. To approach these requirements, we have introduced an enzymatic assay for the inhibition of patient-derived recombinant HIV protease by therapeutical PI using HPLC methodology (Gehringer et al, 2003). Here we describe an improved assay comprising recombinant expression of PR from patient-derived RNA/cDNA in E. coli cells and measurement of the enzymatic activity with a fluorogenic substrate in a microtiter plate-based high-throughput system.…”