enzyme 2  (Sino Biological)

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

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Diagram of the RBD substitutions that distinguish Omicron BA.2.86 from Wuhan-Hu-1 (top), Omicron KP.3.1.1 from BA.2.86 (middle), and Omicron LP.8.1 from BA.2.86 (bottom). Dashed lines show propagation of BA.2.86 changes to KP.3.1.1 and LP.8.1, and italicized mutation in LP.8.1 (H445R) indicates a secondary substitution at a site that previously changed from Wuhan-Hu-1 to BA.2.86. Wuhan-Hu-1 reference spike numbering is used throughout the manuscript. ( B-D ) Quality control of the KP.3.1.1 and LP.8.1 RBD site-saturation mutagenesis library as assessed by PacBio sequencing, illustrating the distribution of number of amino acid mutations per barcoded variant (B), the average number of mutations of each type across library variants (C), and the distribution of mutations across sites in the RBD over all variants (D). ( E, F ) Representative FACS gates used to sort RBD + singlet cells for ACE2 binding (E) and singlet cells for RBD expression (F), which is followed by high-throughput sequencing of cells sorted into each bin. ( G, H ) Correlation in per-mutant deep mutational scanning measurements between independently barcoded replicate libraries for ACE2-binding affinity (G) and RBD expression (H) experiments. Red dash indicates the 1:1 line.

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Mutagenesis, Control, PacBio Sequencing, Variant Assay, Binding Assay, Expressing, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Heatmap illustrating the impacts of all mutations in the KP.3.1.1 and LP.8.1 RBD on ACE2-binding affinity as determined from FACS-seq experiments with yeast-displayed RBD mutant libraries. ACE2 contact residues (black squares, bottom) defined as RBD residues with non-hydrogen atoms <5Å from ACE2 in the BA.2.86 RBD structure (PDB 8QSQ ). Raw data available in Supplemental Data 1. ( B ) Deep mutational scanning data from (A) mapped to the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ ), illustrating the average effect of mutations at a site (left), and the maximal effect of any mutation at a site (right). Sites of interest are labeled. ACE2 (key motifs only) is shown as transparent gray cartoon. ( C ) Heatmap illustrating the impacts of all mutations in the KP.3.1.1 and LP.8.1 RBD on yeast-surface expression levels, a proxy for folding and expression efficiency. Raw data available in Supplemental Data 1. An interactive version of these heatmaps alongside previously assayed SARS-CoV-2 variant RBDs is available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-KP3-LP8/RBD-heatmaps/ .

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Binding Assay, Mutagenesis, Labeling, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Correlation between KP.3.1.1 mutant effects on ACE2 binding in the pseudovirus whole-spike DMS study of Dadonaite et al. (change in potency of pseudoviral neutralization via soluble ACE2 protein) versus the yeast-display RBD DMS study presented here (change in binding affinity from FACS-seq titration). Correlations are separated based on residue distance from the ACE2 interface in the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ ). Mutations whose effect on spike-mediated pseudoviral entry was < -2 units per the assay of Dadonaite et al. were not included because it is difficult to reliably measure soluble-ACE2-inhibition of entry when entry is poor. ( B ) Structural context of residues F374 and H505 in the spike trimer (PDB 9ELH ). Center, overall top-down view of the spike trimer with two RBDs in the “down” and one in the “up” conformation. Left, zoom-in on the down-down interface of RBD packing; right, zoom-in on the up-down interface of RBD packing.

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Mutagenesis, Binding Assay, Neutralization, Titration, Residue, Inhibition

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Epistatic shift in the effects of mutations on ACE2 binding at each RBD position as measured in the Wuhan-Hu-1 (previously reported in ), KP.3.1.1 or LP.8.1 background compared to those previously measured in Omicron BA.2.86 (previously reported in ). ( B ) Mutation-level plots of epistatic shifts between BA.2.86 and KP.3.1.1 or LP.8.1 at sites of interest. Each scatterplot shows the measured ACE2-binding affinity of each amino acid (plotting character, – indicates deletion character) in the BA.2.86 versus KP.3.1.1 or LP.8.1 backgrounds. Red dashed lines mark the respective wildtype RBD affinities on each axis, and the gray dashed line indicates the additive (non-epistatic) expectation. Interactive plots enabling the comparison of all SARS-CoV-2 variants and scatterplots for all RBD sites are available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-KP3-LP8/epistatic-shifts/ .

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Binding Assay, Mutagenesis, Comparison