Vicriviroc Resistance Decay and Relative Replicative Fitness in HIV-1 Clinical Isolates under Sequential Drug Selection Pressures

Abstract

We previously described an HIV-1-infected individual who developed resistance to vicriviroc (VCV), an investigational CCR5 antagonist, during 28 weeks of therapy (Tsibris AM et al., J. Virol. 82: 8210–8214, 2008). To investigate the decay of VCV resistance mutations, a standard clonal analysis of full-length env (gp160) was performed on plasma HIV-1 samples obtained at week 28 (the time of VCV discontinuation) and at three subsequent time points (weeks 30, 42, and 48). During 132 days, VCV-resistant HIV-1 was replaced by VCV-sensitive viruses whose V3 loop sequences differed from the dominant pretreatment forms. A deep-sequencing analysis showed that the week 48 VCV-sensitive V3 loop form emerged from a preexisting viral variant. Enfuvirtide was added to the antiretroviral regimen at week 30; by week 48, enfuvirtide treatment selected for either the G36D or N43D HR-1 mutation. Growth competition experiments demonstrated that viruses incorporating the dominant week 28 VCV-resistant env were less fit than week 0 viruses in the absence of VCV but more fit than week 48 viruses. This week 48 fitness deficit persisted when G36D was corrected by either site-directed mutagenesis or week 48 gp41 domain swapping. The correction of N43D, in contrast, restored fitness relative to that of week 28, but not week 0, viruses. Virus entry kinetics correlated with observed fitness differences; the slower entry of enfuvirtide-resistant viruses corrected to wild-type rates in the presence of enfuvirtide. These findings suggest that while VCV and enfuvirtide select for resistance mutations in only one env subunit, gp120 and gp41 coevolve to maximize viral fitness under sequential drug selection pressures.

Resistance to N-peptide fusion inhibitors correlates with thermodynamic stability of the gp41 six-helix bundle but not HIV entry kinetics

“…Florescence changes were monitored over time using a 409 nm excitation and both a 460 nm (blue) and 528 nm (green) emission detection. We measured fusion for 100 minutes to capture maximum fusion, which was comparable to time points reported by others using similar assays [ 56 , 58 ]. We also noticed that control pseudoviruses without Env produced a time-dependent increase in background signal as incubation periods approach 100 min (Additional file 1 : Figure S1).…”

“…We next assessed whether increased 6HB stability affected the rate of virus entry, since entry kinetics has been linked to susceptibility to inhibition by peptide fusion inhibitors [ 50 - 52 ]. To measure entry kinetics, we recorded fusion in real time using pseudoviruses that incorporated β-lactamase-Vpr enzyme, which cleaves a fluorescent substrate in the cytoplasm of target cells after virus entry [ 55 - 58 ]. This assay showed that five of six Envs selected in the resistance cultures conferred significantly slower (C1-C1, C2-C2, C6-C6, C4-C4, C5-C5) entry kinetics relative to WT (Figure 3 A-B; Table 2 ).…”

Escape from Human Immunodeficiency Virus Type 1 (HIV-1) Entry Inhibitors

“…Deep sequencing of VCV-treated patients showed rapid expansion of V3 sequence diversity and minor variants during treatment, indicating that variants that are initially less than 1% of the quasispecies can be clinically relevant [198]. Furthermore, co-evolution between gp120 and gp41 may help to maximize fitness and entry kinetics [199]. The quasispecies, therefore, provides a ready source of variants for sampling and finding an efficient pathway for resistance.…”

Exposure to Entry Inhibitors Alters HIV Infectiousness and Sensitivity to Broadly Neutralizing Monoclonal Antibodies

“…6) and changes in entry efficiency. [7][8][9][10][11] Selective pressure on Env derives primarily from the humoral immune response. Neutralizing antibodies are those that can prevent virus infection of a target cell.…”