- Authors: Sokal A. et al.
- Year: 2021
- Journal: Immunity S1074-7613 00396-4
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Plasmids coding for recombinant proteins were transiently transfected in Expi293F cells using FectoPRO, according to the manufacturer’s instructions. Cells were incubated at 37C for 5 days and then the culture was centrifuged and the supernatant was concentrated. Proteins were purified from the supernatant by affinity chromatography using StrepTactin columns (IBA) (SARS-CoV-2 S) or HisTrapTM Excel columns (Cytiva) (SARS-CoV-2 RBD). A final step of size-exclusion chromatography (SEC) in PBS was also performed, using either a Superose6 10/300 column (Cytiva) for the SARS-CoV-2 S, or a Superdex200 10/300 (Cytiva) for the SARS-CoV-2 RBD.
In addition to serum immunoglobulins, memory B cell (MBC) generation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is another layer of immune protection, but the quality of MBC responses in naive and coronavirus disease 2019 (COVID-19)-recovered individuals after vaccination remains ill defined. We studied longitudinal cohorts of naive and disease-recovered individuals for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of antibody repertoires, affinity, and neutralization against variants of concern (VOCs) using unbiased cultures of 2,452 MBCs. Upon boosting, the MBC pool of recovered individuals expanded selectively, matured further, and harbored potent neutralizers against VOCs. Although naive individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity toward multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs.