Odn2006

Curated compound collections from the University of Kansas High Throughput Screening laboratory which include Life Chemicals (15,040), ChemBridge (43,736), ChemDiv (56,232), Selleck Bioactives (1649), TimTec (5000), and FDA Repurposed Library (2,286) were used. Compound transfers from source (80 nL of 10 mM stocks) to assay plates were performed using an Echo 550 acoustic liquid handler (Labcyte, Sunnyvale, CA). For most libraries, a target final concentration of 10 μM of compound (in a final volume of 80 μL for the multiplexed reporter gene-based assay described below) was achieved; the FDA Repurposed Library compounds were plated to obtain final concentrations of 2.5 μM. Assay plates were hermetically sealed and stored at -80°C until used. AmpB and nystatin were purchased from Sigma-Aldrich (St. Louis, MO). Synthetic MPLA, lipoteichoic acid (LTA) from S. aureus, PAM₂CSK₄, Poly(I:C), ultrapure LPS from E. coli K12, flagellin from S. typhimurium, ODN-2006 (Vaccigrade), C12-iE-DAP, and Murabutide were procured from InvivoGen (San Diego, CA). The structures of small molecule PRR agonists synthesized by us are shown in S1 Fig. Responses to a variety of TLR and NLR agonists were first examined using THP1-Blue^™ NF-κB reporter cells (InvivoGen, San Diego, CA, Fig 1).

PBMCs were isolated from heparinized blood of four COVID-19 convalescent patients (SARS048, SARS047, SARS044, SARS050) by standard Ficoll density gradient centrifugation using Lymphoprep (Technoclone). EBV-transformed lymphoblastoid B cell lines (EBV⁺ B cells) were generated by supplementing PBMCs with infectious marmoset P95-8 supernatant (ATCC) plus 200 ng/ml cyclosporin A (Sandimmune) in the presence of ODN2006 (1 μg/ml; InvivoGen) at 37°C in 5% CO₂. After 10 days of incubation, cells were replated 1:2 in fresh medium and further cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and 100 μg/ml gentamicin sulfate.

Groups of 6- to 8-week-old female C57BL/6 mice (n = 16 per group; Charles River) were immunized subcutaneously on days 0, 21, and 35 with the following antigens: CPS-CRM197 (2.5 μg/dose of CPS as a conjugate), Hcp1 (5 μg/dose), TssM (5 μg/dose), CPS-CRM197 plus Hcp1, or CPS-CRM197 plus TssM. All antigens were formulated in tissue culture-grade PBS (pH 7.2; Gibco) with Alhydrogel 2% (500-μg/dose; Brenntag) and CpG (20-μg/dose; ODN 2006; Invivogen) as the adjuvant system. Mice immunized with adjuvant only served as controls.

HEK293T cells were transfected with an IFN-β luciferase reporter, pRL-CMV (10 ng; Promega) Renilla luciferase control reporter, pcDNA 3.1 and indicated vectors and 24 h later dual luciferase assays (DLAs) were performed as per published protocols^{60 (link),61 (link)}. For CpG response assays, TLR9-HEK239s (Invivogen) were transfected using lipofectamine with indicated vectors, IFNβ-luciferase and renilla reporters before being stimulated after 24 h with 5 µg/ml CpG (ODN 2006, InvivoGen). The Burkitt’s lymphoma cell line Ramos was nucleofected with the IFN-β luciferase reporter and pRL-CMV using the NEON transfection system (ThermoFisher Scientific) according to the manufacturer’s instructions.

Whole venous blood was collected into tubes containing 1.8 mg/ml K₂EDTA (Becton Dickinson, Oxford, UK). PBMCs were isolated using Ficoll-plaque gradients (Cedarlane, Burlington, ON, Canada) as previously described [17 (link)]. Monocytes were isolated from PBMCs using CD14⁺ selection beads (Miltenyi Biotec, Bisley, UK) as per the manufacturer’s instructions before being cultured in RPMI1640 medium supplemented with 5% (v/v) fetal calf serum (PAN-Biotec, Aidenbach, Germany) and 1% (v/v) penicillin–streptomycin solution (PAA, Pasching, Austria). Cells were incubated with or without 100 ng/ml PAM₃CSK₄ (PAM3) or 10 ng/ml flagellin (Axxora, Exeter, UK), 1 ng/ml FSL-1, 10 ng/ml lipopolysaccharide (LPS), 2 μg/ml resiquimod (R-848), 2 μM ODN2006 or 2 μM ODN2216 (Invivogen, Toulouse, France) for 18 h at 37°C, 5% CO₂. The concentrations of the TLR ligands were previously determined by titrating each ligand to determine the threshold for maximal activation.