Single-cell RNA and B cell receptor profiling of B cell response to SARS-CoV-2 vaccination
Single-cell RNA and B cell receptor profiling of B cell response to SARS-CoV-2 vaccination
Healthy adults were immunized with the mRNA-1273 vaccine formulating mRNA molecules encoding for the full length of the spike (S) protein of the original SARS-CoV-2 strain, during the spring of 2021 as part of the national (United Kingdom) vaccination program. Informed consent was asked of all participants prior to the start of the study. Participants were surveyed for previously known SARS-CoV-2 infection and other relevant (immune-related) co-morbidities at the start and at the end of the study. Whole blood was collected in sodium heparin tubes (455051, Greiner Bio-One). Non-coagulated whole blood from heparin tubes was diluted 1:1 in PBS (14040-091, Gibco) with 2% heat-inactivated fetal bovine serum (FBS, FCS-SA/500-22512, Labtech). Density gradient centrifugation using SepMate tubes (85450, STEMCELL Technologies) was used to isolate peripheral blood mononuclear cells (PBMCs) according to the manufacturer's instructions. PBMCs were counted and viability estimated using a trypan blue exclusion assay. Cells were then resuspended at 10^7 viable cells per ml in FBS containing 10% DMSO (20688, ThermoScientific) and stored in liquid nitrogen. Vaccine-derived antigen-specific B cells were identified by tagging them with the subunit 1 (S1) of the spike protein (S) of the ancestral SARS-CoV-2 and the RBD (M6 timepoint only included S1 tagging). Biotin-conjugated S1 was coupled with two fluorochrome/oligomer dual-labelled streptavidin to form two different S1-fluorochome/oligomer conjugates. In this way, antigen-specific B cells were sorted via flow cytometry assisted sorting (FACS) with nucleotide oligomers to be sequenced as feature barcodes for posterior bioinformatic identification of antigen-specific B cells. For timepoints D-1, D+5, D+9, D+12, W8 and W10 a pool of all S1+ B cells, 60% S1- B cells, 20% CD19- lymphocytes, 20% innate immune cells was performed. For M6 samples, a pool of all S1+ B cells with the addition of S1- B cells up to 100% was performed. All samples were run in multiple 10X reactions lanes for 5000 cells in each reaction. Cells were centrifuged (500g 5 minutes at 4°C) and resuspended in PBS with non-acetylated BSA to the desired concentration and run on the 10X chromium controller utilizing the Chromium Next GEM Single cell 5' reagent kit v2 (Dual Index) (document number: CG00030 Rev F) producing the GEX, VDJ sequencing and cell surface protein libraries according to the manufacturer's instructions. To generate feature barcode libraries tagging antigen-specific B cells for each timepoint, S1-specific B cells were tagged using Totalseq™ streptavidin-PE (405261, Biolegend) and Totalseq™ streptavidin-APC (405283, Biolegend) separately. RBD-specific B cells were tagged using Totalseq™ streptavidin-oligomer (405271, Biolegend). For timepoints before M6, cells from the same donors but collected from different timepoints were multiplexed using TotalSeq hashtags (Biolegend 294661, 394663, 394665, 394667, 394669, 394671). For the M6 timepoint, cells from different donors were multiplexed using the same TotalSeq hashtags (Biolegend) listed above for multiplexing. Hashtagged libraries were generated according to 10X Genomics instructions. Libraries were labelled 'A', 'B', etc. for cells run on different reaction lanes on the 10X Chromium controller.