Resistance-related mutations in the HIV-1 protease gene of patients treated for 1 year with the protease inhibitor ritonavir (ABT-538)

Objective: To define genotypic and phenotypic resistance patterns following prolonged therapy with the protease inhibitor ritonavir (ABT-538). Design: Seven HIV-1-infected patients, all but one previously treated with dideoxynucleoside analogues (zidovudine, didanosine, zalcitabine), were treated for 1 year with ritonavir. Methods: Direct solid-phase sequencing of the protease gene starting from plasma derived viral RNA followed by comparison to phenotypic drug resistance data. Results: The most frequent amino-acid substitutions occurring upon administration of the protease inhibitor were V(82)A/F (substrate binding site), (IV)-V-54 (flap region), A(71)V and L(10)I. Additional mutations found in more than one patient were (IV)-V-15, M(36)I, (IV)-V-84 and I(93)L. Mutation L(63)P was found both in pre- and post-ritonavir samples. Phenotypic drug resistance assays confirmed resistance to ritonavir in post-treatment samples (similar to 170-fold) and showed cross-resistance to indinavir (similar to 30-fold) and partially to saquinavir (similar to fivefold). At 1 year of treatment, one patient without known resistance-associated mutations in the protease gene still showed a substantial rise in CD4 cell count accompanied by a more than 2.4 log decrease in RNA viral load. However, at week 78, mutations R(8)Q, E(34)K, R(57)K, L(63)P and (IV)-V-84 were detected and the treatment benefit was partially lost. Conclusions: Long-term treatment with ritonavir is associated with the emergence of multiple mutations in the HIV-1 protease gene. The mutations L(10)I, (IV)-V-54, L(63)P, A(71)V, V(82)A/F and (IV)-V-84 correspond to known drug-resistance mutations for ritonavir and other protease inhibitors. Phenotypic resistance to ritonavir was detected in a majority of ritonavir-treated patients at 1 year of treatment. In addition, long-term ritonavir treatment selects for cross-resistance to the protease inhibitors indinavir and saquinavir. This argues against sequential therapy with several protease inhibitors. Delayed resistance in one patient was accompanied with a prolonged increase in CD4 cell count and decrease in viral load suggesting a temporary benefit of treatment.