Background:
Lenacapavir (LEN) is a first-in-class, long-acting capsid (CA) inhibitor for the treatment and prevention of HIV-1 infection. While LEN has shown full potency against different HIV-1 subtypes, it remains unclear how viral diversity encountered in the clinic may impact its efficacy. Herein we analyzed HIV-1 CA sequence diversity to identify natural polymorphisms within the LEN binding site and assessed each for their impact on viral fitness and susceptibility to LEN.
Methods:
CA binding site residues within a given radius of LEN were identified in Pymol. HIV-1 CA sequences from public (N=9232) and Gilead trial datasets (N=825) were analyzed for naturally occurring binding site polymorphisms across subtypes A1, B, C, D, F1, G, CRF01_AE and CRF02_AG. Site-directed mutants encompassing CA polymorphisms with a > 0.5% prevalence were expressed as single-cycle NL4.3-based reporter viruses. Infectivity and antiviral EC50 values for WT and mutant viruses were determined in MT-4 cells.
Results:
Of the 25 CA amino acids identified within a 5Å radius of LEN at its binding site, 10 (40%) were completely invariant among the >10K unique HIV-1 CA sequences analyzed. Half (5/10) of these conserved residues (M66, Q67, K70, N74 and A105) matched those previously associated with LEN resistance when mutated. Among each of the remaining 15 LEN binding site residues, at least 1 variant was identified across the 8 subtypes evaluated, with codons S41, Q50, T54 and N183 being the most variable with 6, 8, 7 and 9 substitutions detected, respectively. Site-directed HIV-1 reporter viruses encompassing all of the observed CA variants (n=48) were produced and evaluated for infectivity and drug susceptibility in MT-4 cells. Approximately half (25/48) of these mutants showed impaired infectivity (<50%, range 0.006 – 47%) relative to the WT, with 6 (I37Y, Q50P, N53K, T54Y, I73F, R173K) being so severely impaired that it prevented LEN resistance profiling. Of the remaining 42 CA variants, 39 (93%) remained fully susceptible to LEN (FC=0.6-2.4). Three variants (Q50E, L56V and N57H), with prevalence of ~0.5% in a single subtype (C or D) and impaired infectivity (0.6-25% of WT), showed reduced susceptibility to LEN relative to WT (FC=3.1, 72 and 4890, respectively).
Conclusions:
With few exceptions, our mutant HIV panel comprising rare naturally occurring LEN binding site variants in CA remained fully susceptible to LEN, suggesting that the existing natural viral diversity should minimally impact LEN efficacy in the clinic.