Britelite plus kit

The infectivity and neutralization assays were performed as previously described (41 (link)). Briefly, to measure infectivity, each virus stock was diluted to a concentration of 80 ng Gag-p24/ml and 2-fold serial dilutions were performed eight times, in triplicate, in a 96-well plate. One hundred microliters of each diluted virus stock was transferred to a 96-well plate containing 10,000 TZM-bl cells per well and stored in a 37°C incubator with 5% CO₂. After 48 h, viral infectivity per nanogram of Gag-p24 was determined based on luciferase activity using a Britelite plus kit (PerkinElmer). The neutralization assay was performed similarly, except that a single concentration of virus was used that gave results corresponding to approximately 150,000 relative light units (RLU) after 48 h. Each monoclonal antibody was tested at a primary concentration of 50 µg/ml and subjected to 5-fold titrations seven times. The antibody and virus were incubated together for 1 h prior to the addition of TZM-bl cells. The panel of antibodies tested in the neutralization assays recognized the CD4 binding site (3BNC117, VRC01, VRC07-523, N6), V1/V2-glycans (PG9, PG16, PGDM1400, CAP256-VRC26.25), V3-glycans (10-1074, PGT121, PGT128), gp120/41 (8ANC195), the membrane-proximal external region of gp41 (10E8, 4E10, 2F5), the CD4-induced (CD4i) binding site in gp120 (17b), and V3 (447-52D).

Duplicates of five-fold serial dilutions of the four polymers were tested in human lung tissue (HLT) cells using at least three different donors. HLT cells were added at a density of 300,000 cells/well and incubated with the compounds for 1 h before infection. Then, a MOI of 0.1 of the VSV^*ΔG(Luc)-S virus was added to the wells, and plates were spinoculated at 1,200 g and 37°C for 2 h. After the infection, fresh RPMI medium was added to the wells and cell suspensions were transferred into a 96-well flat-bottom plate. Cells were then cultured overnight at 37°C in a 5% CO₂ incubator. Each plate contained the following controls: no cells (background control), cells treated with medium (mock infection), cells infected but untreated (infection control) and cells infected and treated with the drug camostat mesylate (S2874, Sigma) as a positive control (Grau-Expósito et al., 2022 (link)). After 20 h, cells were incubated with Britelite plus reagent (Britelite plus kit; PerkinElmer) and then transferred to an opaque black plate. Luminescence was immediately recorded by a luminescence plate reader (LUMIstar Omega). In parallel, drug cytotoxicity was monitored by luminescence. To evaluate cytotoxicity, the CellTiter-Glo Luminescent kit (Promega), was used. Data were normalized to the mock-infected control, after which EC₅₀ and CC₅₀ values were calculated.

PBMCs were obtained from HIV-1-infected patients by Ficoll-Paque density gradient centrifugation and cryopreserved in liquid nitrogen. PBMCs from healthy donors were obtained anonymously from the BST (Banc de Sang I Teixits, Barcelona, Spain) and isolated as described above.
The human latently infected cell line J-Lat (clone 9.2) was obtained through the NIH AIDS Reagent Program from Eric Verdin (59 (link)); grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.); and maintained at 37°C in a 5% CO₂ incubator. The T-lymphoblastoid MOLT-4 CCR5+ cell line (obtained through the NIH AIDS Reagent Program from Masanori Baba, Hiroshi Miyake, and Yuji Iizawa) (60 (link)) was cultured in R10 (RPMI medium with 10% FBS) containing 1 mg/ml G-418.
The plasmid encoding HIV-1 strain NL4.3 (pNL4.3) was obtained through the NIH AIDS Reagent Program from Malcom Martin. Viral stocks were generated by transfection of 293T cells with pNL4.3, and the resulting viral particles were titrated in TZMbl cells by enzyme luminescence assay (britelite plus kit; PerkinElmer) as described previously (61 (link)).