The dose escalation selection with TFV commenced at a drug concentration of 5 μM, approximately 1.4-fold above the EC50 of TFV (Fig. 1C). After 145 days in culture, at passage 5 (P5, 80 μM TFV), the K65R mutation was observed. At the following passage (P6, 160 μM TFV), the Y115F mutation in RT was detected in addition to K65R, and the selection was terminated after 173 days. Clonal sequencing was performed on the final viral pool from P6, and K65R and Y115F, as well as viral quasispecies, were observed in 100% of the 38 of viral clones analyzed, possibly the result of a founder effect in which a new population is established from the survival of a few resistant viral variants (Table 2).
Viral pools from the dose escalation selection with EVG contained IN mutations at the E92 residue. The P10 viral pool containing an E92G/Q mixture had moderately reduced susceptibility to both EVG and RAL of 5.3- and 2.2-fold, respectively. The E92G site-directed mutant displayed 3.7-fold reduced susceptibility to EVG and no cross-resistance to RAL. The E92Q mutant was more resistant to EVG with 16-fold reduced susceptibility and low-level cross-resistance to RAL (2.5-fold). HIV-2 viruses with E92G/Q remained fully susceptible to FTC, TFV, AZT, and DRV (fold change compared with wild-type of <2). The S147N mutation was observed at low-level in one breakthrough selection. Despite multiple attempts, the S147N mutant was unable to replicate in cell culture and thus yielded no phenotypic data.
Both dose escalation and breakthrough selections with FTC resulted in the development of M184V/I mutations in RT. The P9 viral pool with M184I was highly resistant to FTC with 78-fold reduced susceptibility. The M184I site-directed mutant yielded 34-fold reduced susceptibility to FTC. The M184V site-directed mutant was also highly resistant to FTC, with >1000-fold reduced susceptibility. HIV-2 viruses with M184V/I had no cross-resistance to the INSTIs, DRV, or the other NRTIs tested.
Selection with TFV led to emergence of the K65R mutation in RT. The P5 viral pool of the TFV dose escalation selection containing K65R had 7.1-fold reduced susceptibility to TFV and 25-fold reduced susceptibility to FTC. The K65R site-directed mutation conferred moderate resistance to both TFV and FTC with 2.2- and 9.1-fold reduced susceptibilities, respectively. The TFV P6 pool contained K65R and Y115F. The Y115F single mutant was fully susceptible to all drugs tested. However, the virus with Y115F and K65R together was more resistant to both FTC and TFV, with 36- and 5.4-fold reduced susceptibilities, respectively. The HIV-2ROD9 K65R + Y115F double mutant also showed minor cross-resistance to AZT with a 2-fold increase in EC50; however, this was not statistically significant. The K65R, Y115F, and K65R + Y115F mutants all remained fully susceptible to EVG, RAL, and DRV.
The dose escalation selection with EVG led to the development of a predominant E92G population and a minor E92Q subpopulation in HIV-2 IN. E92Q is a well-characterized EVG primary resistance mutation selected by HIV-1 in vitro and in vivo.17,28,29 E92G is also associated with EVG resistance in HIV-1 and has previously emerged under in vitro selective pressure from a metabolite of EVG21 and been observed clinically with EVG.21,28 In HIV-1, E92G and E92Q reduce susceptibility to EVG in the range of 30- to 40-fold, and only E92Q shows moderate cross-resistance to RAL,17,21,29,30 similar to the pattern of resistance displayed here by HIV-2ROD9 E92G/Q mutants. Additionally, the E92G/Q mutations are involved with resistance to RAL in HIV-2 infected patients,6,31 and a recent study has shown another HIV-2ROD9 E92Q mutant confers similar fold-changes in RAL and EVG EC50s as reported here.32 An S147N IN mutation developed at low levels in one EVG breakthrough selection after continuous viral replication in the presence of EVG. The S147 residue lies within the flexible loop of the catalytic core domain of IN, and S147G was previously identified as another primary resistance mutation of HIV-1 selected by EVG in vitro and in vivo.17,28 The S147N mutation, however, is not a true EVG breakthrough mutation given that in this experiment, 50× EVG failed to completely suppress the initial infection. Furthermore, the S147N mutation did not replicate in tissue culture when studied in isolation, suggesting that the viral fitness of this variant in the context of HIV-2 is likely very poor.
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