Data from biological replicates in cell\based neutralization assays were assumed to follow normal distribution without normality test owing to the small numbers of replicates

Data from biological replicates in cell\based neutralization assays were assumed to follow normal distribution without normality test owing to the small numbers of replicates. and unfavorable\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. and (Li (Starr potential of FD20 by intraperitoneal injection 6?h before intranasal inoculation of hamsters by live viruses, followed by assessing the effect of FD20 treatment on body weight (Fig?3A), lung titer (Fig?3B and C), nasal titer (Fig?3D and E), and YM-155 HCl pathology in the lung (Fig?3F and G). At 2?days post\inoculation (d.p.i.), infected hamsters displayed reduced activity and began to progressively lose weight throughout the study (up YM-155 HCl to 15% reduction in untreated animals). There was a significant reduction in excess weight loss associated with FD20 treatment compared to PBS at 4 d.p.i. (Fig?3A), as well as a small YM-155 HCl but significant reduction in viral RNA in the lungs of hamsters treated with FD20 (5?mg doses; 62C75?mg/kg) compared to those that were untreated (maturation to improve potency. FD20 engages RBD at a surface distal to RBM To characterize FD20\RBD interactions, we solved the crystal structure of the scFD20\RBD complex at 3.1?? (Fig?4A and Table?1). The structure was refined to 1 1 2 1Cell sizes (?)206.96 57.93 47.21 ()90.0 100.43 90.0Wavelength (?)0.97915Resolution (?)46.43C3.13 (3.24C3.13) a (2020) predicted antibody epitopes on RBD based on the tolerance to mutation regarding expression (structural) and ACE2\binding affinity (functional) using a deep mutational scanning approach. The predicted ideal epitope (Fig?4E and G), surrounding Glu465, consists of 10 residues, 8 of which are surprisingly contained in the FD20 epitope (12 residues) (Fig?4E). The two exceptions, Arg454 and Asp467, are also in proximity (Fig?4G). Because functional and structural constraints are known to correlate with conservation, the analysis reinforces the idea that this FD20 epitope is usually less likely to mutate. Indeed, the mutation frequencies of the FD20 epitope residues from 1.5?million deposited sequences (Elbe & Buckland\Merrett, 2017) ( were relatively low. Thus, there is only one mutation reported for Arg355 (R355T) (as of June 9, 2021), and the mutation frequencies (per million) of the rest residues were below 70, with the exception for Asn354 which was 391 FUT4 (Fig?EV1). Open in a separate window Physique EV1 Uneven distribution of naturally occurring mutants in RBD YM-155 HCl The distribution of naturally occurring mutants in RBD viewed at different angles. Residues are color\coded according to their mutation frequency using the color scheme on the right. The FD20 epitopes are labeled as C spheres which are color\coded based on their conversation type with FD20 (by side chain or by the main chain). Details of the mutations of the FD20 epitope residues. To further test whether the naturally occurring mutations impact FD20s binding and neutralizing activity, we have designed a panel of mutants as outlined in Table?2 using the following criteria. For each epitope residue, the most frequent mutation was chosen unless the substitution was conservative, in which case amino acid with the most dramatic changes was selected. For example, Arg466 was mutated to isoleucine instead of lysine. In addition, the charge of Lys462 and Glu465 were either eliminated by alanine mutation (not naturally occurring) or reversed by the mutations K462E and E465K (found in the database with low frequencies) (Fig?EV1B), respectively; such mutations were expected to weaken the FD20\RBD binding because both residues provided intermolecular salt bridges (Fig?4B). Finally, the epitope\proximal residue Asp467 was mutated to tyrosine to test its structural/functional constraint. The mutations were launched to YM-155 HCl RBD for binding assay separately with ACE2 (Fig?EV2A) and FD20 (Fig?EV2B), and to S protein in SARS\CoV\2?pp for infectivity (Fig?EV2C) and neutralization assay (Fig?EV2D). In addition, the expression level and processing of S (whether S is usually cleaved to produce S1 and S2) were assessed by Western blotting as quality control for trafficking and maturation (Fig EV2E and F). Table 2 Summary of biophysical and biological characteristics of mutants at the FD20 epitope. (2020); in the case of mutants with comparable infectivity to the wild\type, FD20 remained effective for neutralization, indicating FD20s potential to resist escape mutants. Compared with mAbs.