Pore sized filters

Peptides from surface proteins were obtained by the bacterial “shaving” approach as already described [25 (link),30 (link)], with slight modifications. Briefly, 25 mL of cultures were centrifuged at 3500× g for 10 min at 4 °C, and the pelleted bacteria were washed twice with phosphate-buffered saline (PBS). Cells were resuspended in 0.5 mL PBS/30% sucrose (pH 7.4). Protease “shaving” of resuspended bacteria was performed with 1 µg trypsin (Promega, Madison, WI, USA) for 30 min at 37 °C with top-down agitation within an incubator. The resulting digestion mixtures were centrifuged again at 3500× g for 10 min at 4 °C, and the supernatants, which contained the peptides (i.e., the ‘‘surfome’’ fractions) were filtered using 0.22 µm pore-sized filters (Merck-Millipore, Burlington, MA, USA). Surfomes were re-digested with 0.5 µg trypsin overnight at 37 °C with top-down agitation. Peptides were purified prior to analysis, using Oasis HLB extraction cartridges (Waters, Milford, MA, USA). Peptides were eluted with increasing concentrations of acetonitrile (ACN)/0.1% formic acid, according to the manufacturer’s instructions. Peptide fractions were concentrated with a vacuum concentrator (Eppendorf, Hamburg, Germany), resuspended in 100 µL of 2% ACN/0.1% formic acid, and kept at −20 °C until further analysis.

Vero E6 cells (ATCC-CRL-1586) were cultured as in previously described conditions [87 (link),88 (link),89 (link)] in medium (MEM, Gibco, Gaithersburg, MD, USA) with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37 °C in a 5% CO₂ incubator. We infected Vero E6 with four viral strains genotyped by whole genome next-generation sequencing (NGS) as belonging to: Pangolin lineage B.1.1 [90 (link)] (the first major lineage following the Wuhan genotype that circulated during the first epidemic period in France, designated as “Wuhan”) and three variants: Alpha (B.1.1.7), Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529). Culture supernatants were harvested 24 h post-viral infection and then passed through 0.22 µM pore-sized filters (Merck Millipore, Carrigtwohill, Ireland) to remove cells and debris and obtain viral suspension for experiments.