Background
Lenacapavir (LEN) is a HIV-1 capsid inhibitor approved for heavily-treatment experienced (HTE) people with HIV (PWH). This in vitro study aimed to evaluate LEN susceptibility and genetic barrier in B and non-B subtypes derived from therapy naïve (TN) and HTE PWH.
Methods
Thirty-one NL4-3 based recombinant viruses harbouring clinically derived GAG-PR region were generated from plasma samples collected from TN (n=20) and HTE (n=11) PWH enrolled in the Italian PRESTIGIO registry. LEN IC50 was measured through a TZM-bl cell-based assay and fold-change (FC) values were calculated with respect to the NL4-3 strain. In vitro resistance selection (IVRS) experiments were performed by exposing MT-2 cells infected with recombinant viruses and the NL4-3 control to increasing concentrations of LEN. Cultures were stopped when viral breakthrough (VB) was observed at approximately 100X LEN IC50 (2.56 nM) or after 105 days from the start of IVRS. Sanger sequencing of the p24 coding region was performed at each VB to detect emerging mutations. Statistical analyses were performed with GraphPad Prism version 9.0.0.
Results
None of the viruses harboured known LEN resistance mutations. Baseline susceptibility to LEN was comparable between HTE vs. TN (median FC 0.9 [IQR 0.3-1.6] vs. 2.0 [0.8-3.2], p=0.101, Mann-Whitney test) and between B (n=14) vs. non-B (n=19; 3 each F1 and CRF02_AG, 2 each C, D, CRF01_AE and CRF06_cpx, 1 each A1, A6, G, CRF40_BF, URF D/B) subtypes (median FC 1.1 [0.3-3.0] vs. 1.8 [0.9-3.0], p=0.171). By Kaplan-Meier survival analysis, the time to VB was comparable among HTE vs. TN PWH but not B vs. non-B subtypes at approximately 10X (p=0.284 and p=0.047, respectively, log-rank test) and 100X LEN IC50 (p=0.131 and p=0.026, respectively), with lower time to VB observed with B viruses (figure 1). Known LEN resistance mutations emerged in 27/32 cultures including Q67H/R/K+N74D (n=7), Q67H/K+T107N/S (n=6), N74D (n=5), N74D+T107N/D (n=3), Q67H+K70R+T107N (n=2), Q67H+K70R (n=2), and Q67H (n=2), while in 5 cases no emergent mutations were identified. Non-polymorphic substitutions F169L (with Q67H+T107TN), V86M (with Q67H+K70R) and E213D (with Q67H+T107N) were detected in three distinct cases each.
Conclusions
The in vitro genetic barrier to resistance to LEN was generally low but not affected by long-time exposure to antiretroviral therapy. Viruses harbouring non-B GAG-PR showed delayed breakthrough compared to fully subtype B viruses.