EMERGENCE OF RESISTANT VARIANTS OF HIV IN-VIVO DURING MONOTHERAPY WITH THE PROTEINASE-INHIBITOR SAQUINAVIR

We examined the phenotypic and genotypic properties of virus from the peripheral blood mononuclear cells (PBMC) and plasma of eight HIV-1-in fected asymptomatic patients before and during monotherapy with the pr oteinase inhibitor saquinavir. Susceptibility of primary isolates to d rug was assessed in PBMC culture by deriving IC50 and IC90 values. The observed increases in IC50 and IC90 after approximately one year of t herapy with a dosage of 600 mg tds suggests the presence of virus resi stant to saquinavir in vivo. The magnitude of this altered susceptibil ity ranged from three-fold to in one case 100-fold. In two patients a greater than eight-fold decrease in susceptibility to saquinavir was o bserved. Sequencing of the proteinase genes in viral RNA obtained from patient plasma and/or PBMC was carried out by PCR in parallel with se nsitivity testing. In each case between nine and 12 clones were analys ed. In the two patients from whom virus had greater than eight-fold re duction in susceptibility, a point mutation was observed in the viral proteinase (Leu90--> Met/Ile). Further mutations were observed at resi dues 36, 71 and 84 in these subjects. In a third patient, in whom an e ight-fold increase in HIV IC50 of saquinavir was observed, no mutation s were detected in the proteinase; sequencing of proteinase cleavage s ites in viral gag-pol revealed no significant mutations. In no patient was a Gly48--> Val mutation observed, although this has been associat ed with resistance in vitro. The Leu90--> Met mutation was observed in five subjects, but a greater than eight-fold phenotypic change in ant iviral susceptibility was seen in only two of these. Hence, in vivo, t he Leu90--> Met but not the Gly48--> Val mutation is necessary, but no t sufficient, for phenotypic resistance to saquinavir in HIV.