Background:  In patients harboring PI-resistant HIV-1, the virologic response to treatment changes introducing new PI is often transient, due to the emergence of variants with additional protease mutations and extended cross-resistance. We have investigated the role of preexisting minority virus populations in this evolution.

Methods:  From the ANRS104 "Puzzle 1" study--evaluating salvage by LPV+APV in patients having experienced multiple PI failure--8 patients meeting the following criteria were studied:   treatment with LPV+APV failed at 26 weeks (VL>1.5 log₁₀ copies/mL); protease genotype at 26 weeks had at least 1 new mutation relative to baseline; and phenotypic resistance to at least 1 PI (available for 6 patients) had increased more than 2-fold relative to baseline. Standard protease genotype was available at baseline and at 2, 6, and 26 weeks. To detect minority populations expressing mutations that were not seen by standard genotype at baseline but became dominant at 26 weeks, baseline plasma protease sequences were amplified and cloned. Sequence-selective PCR was used to screen 100 to 300 clones for the presence of mutations L10I/F, L24I, K20R, L33F, I47V, or I54M/V. Negative results for 250 clones excluded with 95% probability the presence of a minority population representing >1%. Positive results were confirmed by sequencing.

Results:   For all patients, 1 to 3 new PI resistance mutations were detected by genotyping at 26 weeks. Of the 19 new mutations detected at 26 weeks, 4 were already present at 2 weeks, 5 appeared at 6 weeks (at least as mixed populations), and the 10 others were detected only at week 26. Minority viral populations expressing the mutations detected by genotyping at 2 weeks were detected at baseline in 3/3 cases tested, and represented 33, 14, and 1% of total clones. Preexisting viral populations expressing the mutations first identified at 6 weeks were detected for 1/5 mutations. Similarly, minority viral populations expressing the mutations first detected by genotyping at 26 weeks were found at baseline in 1/6 cases tested, and represented 1% of total clones.

Conclusions:  In patients failing salvage PI treatment, the evolution of protease resistance can be very rapid, involving emergence of minority viral populations present before the change in therapy. When evolution is slower, the new mutations may either be generated de novo or result from selection of sparse preexisting minority populations.

Keywords: minority populations; evolution; drug resistance