Deep sequencing analysis of HIV‐1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE

Abstract

Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS.

Performance comparison of deep sequencing platforms for detecting HIV‐1 variants in the pol gene

“…The DRMs detected by MiSeq and Sanger sequencing were in complete (100%) agreement for those representing more than 20% of the virus quasispecies, which agreed with the findings of previous studies . Plasma samples from patients with primary HIV‐1 infections had higher virus loads than samples from patients who failed antiretroviral treatment.…”

“…Minority DRMs should be more easily detected in high virus‐load samples as they are present in greater absolute quantities with the limitation of a quasispecies more homogeneous in primary infection. As expected, MiSeq sequencing identified minor mutations not observed by Sanger sequencing because they accounted for less than 20% of the quasispecies . Some DRMs were detected only by one or other of the MiSeq and 454 GS‐FLX sequencing methods; they generally accounted for a small percentage of the virus quasispecies.…”

“…The performances of MiSeq and 454 sequencing technologies for HIV tropism determination were compared . Several studies have used MiSeq sequencing to detect HIV minority drug‐resistant variants and compared it with bulk population sequencing but few have compared it with the 454 GS‐FLX platform . Deep sequencing involves massively parallel sequencing of amplified viral sequences and errors as well as template resampling can occur during amplification and sequencing making it difficult to distinguish true minority variants from sequencing errors.…”

“…Another method to correct for PCR bias and sequencing errors is to use primer IDs, which reduce error rates 30-fold 24,25. The repeatability of the MiSeq protocol for detecting DRMs was good, similar to that of the 454 for less than 20% of the quasispecies 14,16,26. Some DRMs were detected only by one or other of the MiSeq and 454 GS-FLX sequencing methods; they generally accounted for a small percentage of the virus quasispecies.…”

Linked dual-class HIV resistance mutations are associated with treatment failure

“…Some analyses have shown that low-frequency drug resistance mutations (DRMs), especially those conferring resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), are associated with virological failure after initiating combination ART-containing NNRTIs (3)(4)(5)(6)(7)(8)(9)(10)(11). In contrast, other studies have found no association with treatment outcomes when low-frequency resistance mutations are detected that confer resistance to an ARV drug used in the regimen (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). These conflicting results make interpretation of resistance results from newer, more sensitive resistance assays difficult (30).…”