Hepes

MOLT-3 (ATCC, Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI-1640; Corning Cellgro, Manassas, VA, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), HEPES, sodium pyruvate (Wisent Inc., St-Bruno, Québec, Canada), and 5 μg/ml plasmocin (Invivogen, San Diego, CA, USA). Hela LT (39) and HEK293T (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Corning Cellgro, Manassas, VA, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific), nonessential amino acids (NEM) (Corning Cellgro), HEPES, sodium pyruvate (Wisent Inc.), and 5 μg/ml plasmocin (Invivogen). U2OS (osteosarcoma) cells (ATCC) and U2OS-Flp-In TREX (kind gift from Dr. Jakob Nilsson, University of Copenhagen) were cultured in the same medium but supplemented with 10% of Nu Serum (Corning Cellgro) instead of FBS and U2OS-Flp-In TREX were maintained with 5 μg/ml of blasticidin (Invivogen). HHV-6B strain Z29 [79 (link)] was produced by our laboratory, as previously described [21 (link)].

Whole blood or leukapheresis samples were acquired from St. Michael’s Hospital, Unity Health. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation using Ficoll-Paque PLUS (GE Healthcare). PBMC were resuspended in R10 medium consisting of RPMI 1640 (Wisent), 10% heat-inactivated fetal bovine serum (FBS; Wisent), 10 mM HEPES (Wisent), 2 mM l-glutamine (Wisent), and 100 U of penicillin-streptomycin solution (Wisent). PBMC were diluted 1:1 with freezing medium consisting of 20% dimethyl sulfoxide (DMSO; Sigma-Aldrich) in FBS and aliquoted for −150°C storage. PBMC used in the T cell epitope mapping were sent to Scisco Genetics Inc. (Seattle, WA) for HLA typing (Table 3).

The INT-407 (ATCC CCL-6) cells were cultivated in Eagle minimal essential medium (EMEM) (Wisent, St-Bruno, QC, Canada) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Wisent) and 25 mM HEPES (Wisent, St-Bruno, QC, Canada). The gentamicin protection assay described previously was adapted to 96-well plates and performed at a multiplicity of infection (MOI) of 20 [34 (link)]. Bacteria were grown overnight in static condition (low aeration) in LB-NaCl (300 mM) to induce SPI-1 and were added in triplicate. After 90 min, infected cells were washed with phosphate-buffer saline (PBS) and fresh medium supplemented with 50 µg/mL gentamicin was added to kill the extracellular bacteria. Cells were lysed with PBS and 0.1% sodium deoxycholate (PBS-DOC) at 90 min (adhesion), 180 min (invasion), and 18 h (survival) post-infection. Serial dilutions were performed for enumeration of viable colony counts by colony-forming units (CFU/mL). The assay was performed at least three times in triplicate.