Drug Susceptibility Assays

Monogram Biosciences performed susceptibility assays using the PhenoSense HIV-1 Gag/PR assay (hereafter called the Monogram assay), which is a pseudotype-based, single-cycle assay.³⁰ DRV was chosen as the representative PI to test susceptibility of the isolates. Recombinant virus stocks [pseudotyped with amphotropic murine leukemia virus env proteins] were produced by cotransfecting HEK 293T cell cultures with amphotropic murine leukemia virus env and pHIVluc-resistance test vectors. Viral stocks were deposited in 96-well plates containing serial dilutions of PIs spanning an empirically determined range for each drug. Viral stocks were harvested approximately 48 hours after transfection and used to inoculate fresh HEK 293T cell cultures. Replication was monitored by measuring luciferase expression in the infected target HEK 293T cells ∼72 hours after infection.

Drug susceptibility data were determined by plotting the percent inhibition of luciferase activity versus log₁₀ drug concentration.¹⁵ The fold-change in drug susceptibility (FC-IC₅₀) was determined by dividing the drug concentration leading to 50% viral inhibition (IC₅₀) values for the Gag/PR recombinant virus by those of a drug-sensitive reference virus containing the Gag/PR sequences of NL_4-3.

Single-cycle pseudotype–based (hereafter called the single-cycle assay) and multiple-cycle susceptibility assays were performed at Bristol-Myers Squibb. ATV and LPV were chosen as the representative PIs to test susceptibility of the isolates. For the single-cycle assay, 10 μg of full-length pNLRepRuc variant (containing Gag/PR genes from clinical isolates) and 8 μg of plasmid SV-A-MuLV-env were cotransfected into HEK 293T cells in T75 flasks using a calcium precipitation method (Thermo Fisher Scientific). Transfected cells (100 μL) were seeded onto 96-well plates which contained 100 μL of compound dilutions and after ∼30 hours, 100 μL of supernatant (containing newly produced virus) was transferred to freshly cultured HEK 293T cells and maintained for 2 days. Cell-associated Renilla luciferase activity was measured by the addition of EnduRen Live Cell Substrate (Promega). For multiple-cycle assays, MT-2 cells were infected with pooled full-length virus-containing Gag/PR genes from clinical isolates in the pNLRepRuc backbone versus wild-type control virus. Cell–virus mixtures were seeded onto 96-well plates containing serially diluted compounds. After 4 days' incubation at 37°C/5% CO₂, virus growth was determined by measuring the activity of cell-associated Renilla luciferase as described above.

Longitudinal isolates (pre- and post-PI treatment) were obtained from 15 patients receiving PIs as part of their combination ARV therapy regimen for a median (range) of 6 (2.3–11.7) years (Table ​(Table1).1). There were 21 post-PI treatment samples collected while patients were on PI therapy (9 patients had 1 post-treatment sample and 6 patients had 2 post-PI treatment samples). Cloning of Gag/PR amplicons from all 15 patients was performed by Monogram Biosciences. However, only samples from Pts02, 03, 04, 06, 07, 09, 10, 11, 12, 14, and 15 produced pooled clones that could be analyzed for phenotypic sensitivity to GSK3532795 and a clinically relevant PI (DRV). Some pre- or post-PI treatment samples, or both, from Pts01, 05, 06, 08, and 16 yielded a nonreportable result from the Monogram assay but were recloned at Bristol-Myers Squibb and analyzed for phenotypic susceptibility to GSK3532795, BMS MI A and BMS MI B, and 2 clinically relevant PIs (ATV and LPV). For samples in which a potentially elevated GSK3532795 susceptibility was identified by the Monogram assay [changes from baseline (CFB) >3-fold], additional cloning and re-analysis was performed using the single- and multiple-cycle assays (Supplemental Digital Content, Fig. 1 and Table ​Table22 http://links.lww.com/QAI/A974).

Treatment History, Genotype, and Predicted Phenotypic Susceptibility of all Longitudinal Samples

Longitudinal Isolates (Monogram Assay): GSK3532795 and DRV Phenotypic Susceptibility, and Gag Genotype