Abstract
SummaryAlpha-B.1.1.7, Beta-B.1.351, Gamma-P.1, and Delta-B.1.617.2 variants of SARS-CoV-2 express multiple mutations in the spike protein (S). These may alter the antigenic structure of S, causing escape from natural or vaccine-induced immunity. Beta is particularly difficult to neutralize using serum induced by early pandemic SARS-CoV-2 strains and is most antigenically separated from Delta. To understand this, we generated 674 mAbs from Beta-infected individuals and performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor-binding domain (RBD). Two of these RBD-binding mAbs recognize a neutralizing epitope conserved between SARS-CoV-1 and -2, while 18 target mutated residues in Beta: K417N, E484K, and N501Y. There is a major response to N501Y, including a public IgVH4-39 sequence, with E484K and K417N also targeted. Recognition of these key residues underscores why serum from Beta cases poorly neutralizes early pandemic and Delta viruses.
Highlights
T f assays were performed and 5 cases with the highest titres were selected for further study oo (Figure 1A)
As expected, focus reduction was calculated and IC50 (FRNT50) titres were higher for Beta than pr Victoria (Figure 1B). re- To isolate memory B cells PBMC were stained with full length double Strep tagged Beta S and l P IgG+ B cells binding Beta S were single cell sorted (Figure 1C,D)
IgVH and IgVL sequences were na isolated by RT-PCR and full-length heavy chain (HC) and light chain (LC) expressing plasmids ur were created using a Gibson assembly reaction
Summary
T f assays were performed and 5 cases with the highest titres were selected for further study oo (Figure 1A). For these selected cases, as expected, FRNT50 titres were higher for Beta than pr Victoria (an early Wuhan related viral isolate) (Figure 1B). Products were transfected into Jo HEK-293T cells in 96 well plates and supernatants harvested and tested in neutralization assays against Beta virus at a final concentration of 0.1-1 g/ml. Those mAbs achieving >90% neutralization in this initial assay were selected for further study. Most were able to block ACE2 binding to S, there were exceptions: mAb Beta-43, the single NTD binding neutralizing antibody (Figure S1A), extremely potent RBD binding mAb Beta-53 and potent but very weakly RBD-binding mAbs Beta-49 and 50 (Figure 1G,H)
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