Journal: PLOS Pathogens

Article Title: Neutralization of SARS-CoV-2 by IgM-14 via engagement of two distinct spike epitopes

doi: 10.1371/journal.ppat.1014071

Figure Lengend Snippet: A , Diagram of the process for selecting IgM-14-resistance in VeroE6 cells. Four independent selections (SL1-4) started at passage 1 (P1) and were passaged 6 times. SL4 was aborted at the second passage. B , Spike mutations occurred in P6. EC 50 s of IgM-14 against P6 viruses (SL1-3) are shown. *, intact and deletion sequences observed in the selection. C, Construction of mNG SARS-CoV-2 with spike mutations. D , Neutralization curves of IgM-14 and IgG-14 against WT or mutant mNG SARS-CoV-2. E , Summary of the EC 50 values derived from panel D. F , Plaque morphologies of WT or mutant mNG SARS-CoV-2 on VeroE6 cells. Growth kinetics of G476D ( G ) or F486S ( H ) versus WT mNG SARS-CoV-2 in VeroE6, A549-hACE2, and human airway epithelial (HAE) cultures. Two-way ANOVA with Šidák’s multiple comparison corrections was used for statistical analysis. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. I , Relative expression of RBD mutants to the WT RBD. Error bars indicate variation across three independent experiments. J , BLI analysis of the binding of WT and mutant RBDs to human ACE2. Association rate ( k on ), dissociation rate ( k off ), affinity constant ( K D ), and R 2 values of curve fitting are indicated. K, ELISA analysis of IgM-14 and IgG-14 binding to WT (solid circles), G476D (red circles), and F486S (blue circles) RBDs. Error bars indicate variation across three independent experiments.

Article Snippet: The human ACE2 protein (10108-H08H) was purchased from Sino Biological.

Techniques: Selection, Neutralization, Mutagenesis, Derivative Assay, Comparison, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: Neutralization of SARS-CoV-2 by IgM-14 via engagement of two distinct spike epitopes

doi: 10.1371/journal.ppat.1014071

Figure Lengend Snippet: A, overview of the primary binding site. B , Zoomed-in view of the interaction between HCDR2 and the RBD paperclip motif. Pink dashed lines indicate salt bridges, purple dashed lines show hydrogen bonds. C , Zoomed-in view of the interaction between Fab-14 light chain and RBD residues G476-P479. D , Superposition of Fab-14 and ACE-2 on the same up RBD. The surface of ACE2 and Fab-14 are shown in pink and blue, respectively. E , MM/PBSA analysis of changes in RBD/Fab-14 complex binding free energy changes caused by individual mutations. Data shows the mean ± standard deviations. F , Sequence alignment of the primary binding site between the parental SARS-CoV-2 (USA-WA1/2020) and Omicron variants. The GISAIDs of BA.1, BA.2, BA.3, and JN.1 spikes were EPI_ISL_6640916, EPI_ISL_6795834.2, EPI_ISL_7605591, and EPI_ISL _18237538, respectively.

Article Snippet: The human ACE2 protein (10108-H08H) was purchased from Sino Biological.

Techniques: Binding Assay, Sequencing