Lsr4 laser

Binding of specific antibody binding to cells were determined by LSR4 laser (BD Biosciences) and analyzed using FlowJo (Tree Star). Cells were gated on: FSC-A/SSC-A to exclude cell debris (Figure S1A), FSC-A/FSC-H to select for single cells (Figure S1B), FSC-A/PI to select for live cells (PI-negative population; Figure S1C), FITC/Alexa Fluor 647 (Figures S1D–S1H). Binding is determined by the percentage of GFP-positive S protein-expressing cells that are bound by specific antibody, indicated by the events that are Alexa Fluor 647- and FITC-positive (Gate 2). A sample is defined as positive when the binding is more than mean + 3SD of the healthy controls (n = 22). The thresholds using the healthy control readings is based on the normal-like distribution of the healthy control reading where a mean + 3SD threshold would mean that there is less than a 0.13% chance of a false positive. Receiver Operating Characteristic (ROC) curves were constructed from each of the antibody binding with the healthy controls and SARS-CoV-2 patients as the true negatives and true positives respectively using the pROC library in R version 3.6.4.

The SFB assay was performed according to previously described methods (16 (link), 17 (link)). In brief, cells expressing the spike protein of the ancestral Wuhan strain, Omicron BA.1 and XBB were seeded at 1.5 x 10⁵ cells/well in 96-well V-bottom plates (Thermo Fisher Scientific, Waltham, USA). The cells were incubated with human serum (diluted 1:100 in 10% FBS; HyClone, Chicago, USA), followed by a second incubation with a double stain comprising Alexa Fluor 647-conjugated anti-human IgG (diluted 1:500; Thermo Fisher Scientific) and propidium iodide (PI; diluted 1:2500; Sigma-Aldrich, Burlington, USA). Cells were acquired using an LSR4 laser (BD Biosciences, New Jersey, USA) and analyzed using FlowJo (Tree Star, BD Biosciences). The percentage of GFP-positive spike protein-expressing cells bound by the antibody, indicated by Alexa Fluor 647- and FITC-positive events, was used as an indicator of binding. The assay was performed as two independent experiments, each with technical duplicates. The amount of spike protein expressed on the cell surface was verified by ACE-2-HuFc binding. A subset of age-matched samples was randomly selected and examined for binding antibodies against Omicron BA.1 and XBB (n = 10 per LTRs with double and triple IS regimens and HC).