Profiling Vaccine-Induced Immune Responses

PBMCs were prepared by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Chicago, IL, USA). PBMCs before and after 3^rd vaccination were cryopreserved at -80°. Cells were thawed, washed twice in pre-warmed RPMI1640 medium [containing 0.3 mg/ml glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% FCS and 25 U/ml DNase I (Roche International)] and stained as described (15 (link)). To identify RBD-specific and TT-specific B cells, respectively, recombinant purified RBD (DAGC149, Creative Diagnostics, New York, USA) and TT (peptides & elephants GmbH, Hennigsdorf, Germany) were labeled with either AF647 or AF488. Double positive cells were considered antigen-specific as reported. A blocking experiment using unlabeled RBD or TT respectively in 100-fold concentration was used to ensure specificity of detection as reported (15 (link), 18 (link)). For intracellular staining after T cell stimulation cells were first stained for 30 min with 1:1000 BUV395 Life/Dead (Invitrogen) in PBS, followed by 5 min 2.5 µl Fc Block (Milteny Biotech) in 50 µl resuspended cells. Cells were fixed in LyseFix (Becton Dickinson), permeabilized with FACS Perm II Solution (Becton Dickinson) and intracellularly stained.