Uncovering a conserved vulnerability site in SARS‐CoV‐2 by a human antibody

Abstract

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBMtargeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a K D of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants

“…Three rounds of solution panning were performed with increasingly stringent conditions and an off-selection step to screen high-affinity nanobodies. Subsequent screening using ELISA and fluorescence-detection size exclusion chromatography (FSEC; Li et al, 2021a , b ) identified binders with ELISA signal that is at least three times higher than a control nanobody, as well as the ability to shift the gel filtration peak of fluorescently labeled RBD at 0.5 μM ( Figure 1A ). We identified 28 unique clones as positive clones and we focus on DL28 for this study.…”

“…Due to high demands and limited BSL3 laboratory resources during the continuing outbreak, the neutralization activity of DL28 against the authentic SARS-CoV-2 and the VOCs were not tested in this study. However, accumulating evidence from publications in this field since early 2020 has highlighted a strong correlation between assays using pseudovirus and authentic virus (Bewley et al, 2021 ; Jones et al, 2021 ; Li et al, 2021a , b ). Thus, it is likely that DL28 would neutralize the authentic viruses too.…”

“…According to an antibody survey (Dejnirattisai et al, 2021 ), antibodies that target RBM generally neutralize SARS-CoV-2 with higher activity, and the exact reasons are yet to be discovered. Nevertheless, non-RBM-targeting antibodies like DL28 are attractive candidates for therapeutic cocktails due to their generally broader spectra (Huo et al, 2020b ; Pinto et al, 2020 ; Zhou et al, 2020b ; Li et al, 2021b ).…”

Isolation, characterization, and structure-based engineering of a neutralizing nanobody against SARS-CoV-2

“…Accumulating evidence has suggested a general pattern regarding antibody characteristics and epitope. RBM-targeting antibodies are generally more potent than those targeting the RBD core [ 9 , [63] , [64] , [65] ] but are more susceptible to escape mutants [ 64 , [66] , [67] , [68] , [69] , [70] ]. Consistent with this trend, despite DL4's resistance to the mutations in the Alpha strain, its neutralizing activity is compromised or fully lost against variants with more RBM mutations.…”

“…S assembles into a trimer and is heavily decorated by glycosylation to escape immune surveillance. Suiting the role of molecular recognition, the RBD is relatively less glycosylated and therefore represents a hot spot for neutralizing antibodies [ [5] , [6] , [7] , [8] , [9] , [10] , [11] ] and vaccine development [ [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] ]. Much of the RBD and the receptor-binding motif (RBM, the ACE2-binding surface) are shielded by the N-terminal domain of S1 from adjacent protomers of the S trimer in the so-called more stable “closed conformation”; and such RBDs are referred to as “down”-RBD.…”