Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: A) Binding of IgG and Fab to recombinant RBDs of Sarbecoviruses from different clades by ELISA including SARS-CoV-2 (clade 1b), WIV-1 (clade 1a), Rs4321 (clade 1a), RacCS203 (clade 2), Rc-o319 (clade 5), and RhGB07 (clade 3). Experiments were run in duplicate and repeated twice. A representative assay is shown. Fab binding is shown with the dotted line and IgG binding with the full line. B) Neutralising activity of IgGs against SARS-CoV-2 and variants of concern as well as SARS-CoV-1 and WIV-1. C) IC 50 values for neutralisation against SARS-CoV-2 variants of concern and other sarbecoviruses that utilise human-ACE2. For A–C, the mean value is plotted with error bars showing ± standard deviation (s.d.). D) Ability of IgGs to inhibit the interaction between cell surface human ACE2 and soluble SARS-CoV-2 and WIV-1 RBDs. IgGs were pre-incubated with fluorescently labelled RBD before addition to HeLa-ACE2 cells. The percentage reduction in mean fluorescence intensity is reported. Experiments were performed in duplicate. E) Superimposed 3D structures of the V1WT_06 (dark blue), V1WT_41 (purple), and VA14_26 (red) around WIV-1 RBD (light blue, from PDB 8WLZ). The data underlying this Figure can be found in .

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Incubation, Fluorescence

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: A) A closeup view of the superimposed structure of V1WT_06 bound to WIV-1 RBD on a trimeric spike (from PDB: 8WLZ). A clash with NTD of chain B is observed when V1WT_06 Fab-RBD complex is aligned onto RBD in the up conformation (chain A) in trimeric WIV-1 spike protein. Notably, the binding site is inaccessible in the RBD-down conformation. B) V1WT_06 heavy chain only interaction with WIV-1 RBD (grey). Heavy and light chains are shown with the dark and light blue ribbons, respectively. C) V1WT_06 epitope footprint overlayed with epitope footprints of other site V neutralising antibodies including S2H97, CC25.4, 5817, R1-32, and E465 patch [ , – , ]. SARS-CoV-2 RBD (from PDB: 6m0j) is coloured based on the amino acid conservation across 87 bat sarbecoviruses [ , , , ]. D) Sequence alignment of a panel of bat sarbecovirus RBDs. Contact residues of V1WT_06 mAb on the WIV-1-RBD are indicated with black dots. The colouring scheme describes the percent identity (PID) between the sequences as implemented in Jalview 2.11.4.0. E) Comparison in neutralisation potency of V1WT_06 Fab and IgG against SARS-CoV-2 (D614G), SARS-CoV-1, and WIV-1. Mean values are plotted with error bars showing ± standard deviation (s.d.). The data underlying this Figure can be found in .

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Binding Assay, Sequencing, Comparison, Standard Deviation

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: A) VA14_26 and V1WT_41 antibody footprints overlayed with the epitope footprints of other RBD class 4 neutralising antibodies including CR3022, 10−40, ADI-62113 [ – ]. SARS-CoV-2 RBD (from PDB: 6m0j) is coloured based on the amino acid conservation across 87 bat sarbecoviruses [ , , , ]. B) Superimposed 3D structure depicting the binding of VA14_26 (Red) to WIV-1 RBD (blue) in relation to ACE2 (orange, from PDB: 8WLZ) and CR3022 (pink, from PDB: 6W41). C) Superimposed 3D structure depicting the binding of V1WT_41 (green) to WIV-1 RBD (blue) in relation to ACE2 (orange, from PDB: 8WLZ). D) Sequence alignment of a panel of bat sarbecovirus RBDs. Contact residues for VA14_26 and V1WT_41 are indicated by red and pink dots and outline, respectively. Shared contact residues are shown with yellow dots and outline. E) Sequence alignment of a panel of SARS-CoV-2 variant RBDs. Contact residues for VA14_26 and V1WT_41 are indicated by red and pink dots and outline, respectively. Shared contact residues are shown with yellow dots and outline. The colouring scheme describes the percent identity (PID) between the sequences as implemented in Jalview 2.11.4.0. F) A close-up view of YYDRSG motif in V1WT_41 HCDR3 (green) in contact with WIV-1 RBD (grey), shown in stick representation, renumbered based on alignment to SARS-CoV-2 [ , ]. Presence of G109 breaks the H-bonding pattern in the beta hairpin to create a G1 β-bulge. This further enables D106 side chain to make hydrogen bond with the amides in the protein backbone, thereby stabilising the local structure. Combined this accentuates a right-handed twist found in G1 β-bulge . Twisting of the β-hairpin is thought to contribute towards stabilisation of RBD-Fab interaction in several other public mAbs with YYDRxG motif [ , ].

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Binding Assay, Sequencing, Variant Assay

Journal: PLOS Biology

Article Title: Targeting of conserved spike epitopes enables broad antibody neutralisation of diverse bat sarbecoviruses

doi: 10.1371/journal.pbio.3003882

Figure Lengend Snippet: Neutralisation of sarbecoviruses through entry via different host ACE2s. Neutralisation of A) Rc-o319 and B) RhGB07 was measured against entry via Rh. cornutus ACE2 and Rh. alcyone ACE2, respectively, which were transiently expressed in HEK293T/17 cells. C) Neutralisation of WIV-1 was compared between entry via hu-ACE2 (dotted line and closed symbols) and Rh. alcyone ACE2 (full line and closed symbols). Neutralisation was measured using HEK 293T cells transiently expressing either Human ACE2 or Rh. alcyone ACE2. For A-C, mean values are plotted with error bars showing ± standard deviation (s.d.). D) Ability of IgGs to inhibit the interaction between cell surface ACE2 (including hu-ACE2, Rh. cornutus ACE2 and Rh. alcyone ACE2) and soluble RBD (including SARS-CoV-2, WIV-1, Rs4231, RhBG07, and Rc-o319 RBDs). IgGs were pre-incubated with fluorescently labelled RBD before addition to ACE2 expressing cells. N.B = No binding of IgG to the RBD. The percentage reduction in mean fluorescence intensity is reported. Experiments were performed in duplicate. E) Antibody dependant cellular cytotoxicity (ADCC) of IgGs measured through the surface expression of CD107a on activated NK cells (NK-92 cells expressing CD16) . HIV-1 specific neutralising antibody, PGT128 is used as a negative control . The data underlying this Figure can be found in .

Article Snippet: HEK293T cells were transfected with either Human, Halcyon or Japanese horseshoe bat ACE2 using PEI Max (1 mg/mL, Polysciences) at 3:1, 6:1, and 2:1 ratio (PEI Max:DNA), respectively.

Techniques: Expressing, Standard Deviation, Incubation, Binding Assay, Fluorescence, Negative Control

Journal: PLOS Pathogens

Article Title: Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape

doi: 10.1371/journal.ppat.1014223

Figure Lengend Snippet: A bispecific nanobody, Nanosota-9A/9B-Fc, was generated by fusing Nanosota-9A and Nanosota-9B to a human Fc domain. (A) Efficacy of Nanosota-9A/9B-Fc in neutralizing Omicron pseudoviruses. The assay was performed as in . (B) Efficacy of Nanosota-9A/9B-Fc in neutralizing authentic Omicron in vitro. Each Omicron subvariant infected Vero E6-ACE2-TMPRSS2 cells in the presence of Nanosota-9A/9B-Fc at different concentrations. Infection efficiency was determined by the remaining cell viability after 96 hours (lower viability indicates higher infection). Efficacy for each subvariant is expressed as the IC₅₀, the concentration that reduces virus-induced cytopathic effect by 50% relative to serum-exposed virus controls. Data are mean ± SEM (n = 4). (C) Efficacy of Nanosota-9A/9B-Fc in neutralizing authentic Omicron (JN.1, KP.2, and KP.3) in mice. The experiment followed and , except lung virus titers on day 2 post-infection were measured by TCID₅₀ assay. Treatment groups, n = 5; control groups, n = 4. Comparisons of lung virus titers between the control and treatment groups were performed using an unpaired two-tailed Student’s t-test. **p < 0.01.

Article Snippet: HEK293T and Vero E6-ACE2-TMPRSS2 cells (Vero E6 cells overexpressing human ACE2 and human TMPRSS2) (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Generated, In Vitro, Infection, Concentration Assay, Virus, Control, Two Tailed Test

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 reduces cholesterol abundance in the plasma membrane in general and lipid rafts in particular, and decreases lipid raft area more efficiently than atorvastatin (ATO). Control HEK293T (A) and Calu-3 (B) cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with cholesterol-binding mCherry-conjugated D4H*, the D434S mutant of domain 4 (D4) of C. perfringens theta-toxin. Fluorescence intensities correlating with plasma membrane cholesterol levels of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensity values obtained in n = 9 independent biological replicates, and their average values (± SEM) are plotted in the figure. (C) To examine changes in the cholesterol content of raft and non-raft microdomains of the plasma membrane, control HEK/ACE2 + TMPRSS2 cells and those treated as above were labeled with Alexa Fluor 647-conjugated cholera toxin subunit B (CTX-AF647), a lipid raft marker, and F66. F66 is a fluorescent indicator with spectral properties depending on the cholesterol-dependent local molecular order (dipole potential) of the membrane; therefore, this dye, combined with CTX-AF647, can provide information about the extent of cholesterol reduction separately in raft and non-raft membrane regions. Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show F66 intensities detected in 2 wavelength ranges of emission (“F66 N*” and “F66 T*”), their ratio (“F66 emission ratio” calculated as T*/N* pixel by pixel), and CTX-AF647 intensities. Cell “membrane masks” selected manually in CTX images were segmented using the maxentropy algorithm to CTX-high “rafts” and CTX-low “non-rafts” corresponding to high- and low-intensity regions, respectively, as shown by the representative images. Violin plots were generated from median F66 emission ratio values determined separately for the CTX-high “raft” (D) and CTX-low “non-raft” (E) masks of n = 54 to 73 individual cells, which also display median values with quartiles. (F) Pixelwise distributions of the F66 emission ratio in CTX-high “rafts” and CTX-low “non-rafts” of control cells are displayed. For the quantification of the relative area of lipid rafts, as an alternative definition for raft regions, a threshold value of the F66 emission ratio was determined (green dashed line) and membrane pixels were considered as “F66 raft” and “F66 non-raft” regions when being above and below the threshold, respectively. (G) Violin plots were generated from the relative fraction of F66 raft pixels (“F66 raft area”) of individual cells, which also display median values with quartiles. (H) Representative images show changes in the lateral distribution of the F66 emission ratio on a color-scale image and reduction in the relative F66 raft area induced by 1 μM SH42. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Clinical Proteomics, Membrane, Control, Labeling, Binding Assay, Mutagenesis, Fluorescence, Flow Cytometry, Marker, Generated

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 decreases ACE2 binding of SARS-CoV-2 spike receptor-binding domains (RBDs) more efficiently than ATO. (A) ACE2-expressing HEK/ACE2 + TMPRSS2 and Calu-3 control cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated with the GFP-conjugated RBDs of the Wuhan-Hu-1 strain (WT), Delta, and Omicron BA.1 variants for 4 min. RBDs were applied at 0.2 and 1.0 μg/ml for HEK/ACE2 + TMPRSS2 and Calu-3 cells, respectively. Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. (B) Representative RBD-GFP versus forward-scattered light intensity (FSC) density plots demonstrate decreases in the bound WT RBD-GFP in response to 1 μM SH42 in HEK/ACE2 + TMPRSS2 cells. Dashed lines represent average values of the fluorescence intensity obtained in the displayed representative samples. The average intensities obtained in n = 9 independent biological replicates and normalized to the mean value determined in untreated control samples, and their average values (± SEM) are plotted for WT, Delta, and Omicron BA.1 variants in HEK/ACE2 + TMPRSS2 (C) and Calu-3 (D) cells. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, **** P < 0.0001), while hashes show those between samples treated with ATO and SH42 at identical concentrations ( ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Binding Assay, Expressing, Control, Incubation, Fluorescence, Flow Cytometry

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42-induced reduction in ACE2 binding of WT SARS-CoV-2 spike RBDs negatively correlates with the applied RBD concentration. ACE2-expressing HEK/ACE2 + TMPRSS2 (A) and Calu-3 (B) control cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated with different concentrations of the GFP-conjugated RBDs of the Wuhan-Hu-1 strain (WT) for 4 min. Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The extents of inhibition (calculated as 1 − average of treated/average of control) were determined in n = 9 independent biological replicates, and their average values (± SEM) are plotted as a function of the applied RBD concentration ranging between 0.1 and 5 μg/ml for HEK/ACE2 + TMPRSS2 and between 1 and 10 μg/ml for Calu-3 cells. Asterisks indicate significant differences between samples treated with the lowest versus highest RBD concentrations for each treatment (** P < 0.01, **** P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Binding Assay, Concentration Assay, Expressing, Control, Incubation, Fluorescence, Flow Cytometry, Inhibition

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 inhibits the cellular entry of SARS-CoV-2 spike trimers more efficiently than ATO. Control HEK/ACE2 + TMPRSS2 and Calu-3 cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated for 4 h in the presence of WT, Delta, or Omicron BA.1 SARS-CoV-2 spike trimers conjugated with Alexa Fluor 488 (AF488-trimers) and labeled with F66. (A) Representative orthogonal views of confocal Z-stack images of F66 for the visualization of the plasma membrane and AF488-trimers to estimate entry demonstrate notable trimer accumulation in the intracellular space of untreated control HEK/ACE2 + TMPRSS2 cells. During image analysis, pixels corresponding to plasma membrane and intracellular pixels were segmented based on F66 Z-stack images. Markers were manually placed inside cells (green circles in the grayscale orthogonal view), and a MATLAB implementation of the 3D watershed algorithm identified the intracellular space of cells and their membrane (colored regions and red lines in the orthogonal view in the middle, respectively, and their overlay image displayed on the right). (B) Representative 3D reconstruction images displaying AF488 fluorescence intensities on a green-red color scale above a threshold intensity overlaid on intracellular pixels of individual cells (in transparent blue) demonstrate decreases in the amount of intracellular WT trimers in response to 1 μM SH42. Subsequently, the average fluorescence intensity values emitted by AF488-trimers were calculated exclusively from data of intracellular pixels for individual cells. The average intensities obtained in n = 400 to 600 HEK/ACE2 + TMPRSS2 (C) and Calu-3 (D) cells and normalized to the median value determined in untreated control samples are plotted along with median values with quartiles for WT, Delta, and Omicron BA.1 trimer variants. Asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA. Lognormal functions fitted to normalized mean intracellular AF488-trimer fluorescence intensity histograms of individual HEK/ACE2 + TMPRSS2 (E) and Calu-3 (F) cells also demonstrate the effects of ATO and SH42 on the internalization of WT, Delta, and Omicron BA.1 trimer variants.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Control, Incubation, Labeling, Clinical Proteomics, Membrane, Fluorescence

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 decreases cell surface ACE2 expression and its colocalization with lipid rafts more efficiently than ATO. (A) Control HEK/ACE2 + TMPRSS2 and Calu-3 cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with Alexa Fluor 488-conjugated anti-ACE2 antibodies (AF488-anti-ACE2). Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensities obtained in n = 10 independent biological replicates and normalized to the mean value determined in untreated control samples, and their average values (± SEM) are plotted in the panel. (B) Control cells and those treated as above were labeled with AF488-anti-ACE2 and Alexa Fluor 647-conjugated cholera toxin subunit B (AF647-CTX). Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show AF488-anti-ACE2 and AF647-CTX intensities, and their overlay, while the colocalization of the 2 signals and its changes in response to 1 μM SH42 are displayed in representative dot plots obtained from pixelwise fluorescence intensities. (C) Violin plots were generated from Pearson correlation coefficient values between fluorescence intensities of the 2 applied fluorophores determined from pixelwise data of n = 81 to 90 individual cells, which also display median values with quartiles. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Expressing, Control, Labeling, Fluorescence, Flow Cytometry, Membrane, Generated

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: DHCR24 inhibition by its selective blocker SH42 is a potential novel therapeutic approach to suppress initial membrane-coupled events of SARS-CoV-2 infection. SH42, a novel steroidal highly selective and potent DHCR24 inhibitor, interferes with ACE2 binding of SARS-CoV-2 spike RBDs and cellular uptake of spike proteins. By efficiently decreasing cholesterol levels of the host cell plasma membrane and causing the concomitant disruption of lipid raft microdomains, SH42 decreases cell surface levels of ACE2 and, in addition, reduces raft partitioning of the receptor protein, thereby altering its local microenvironment required for an efficient ACE2-mediated cellular binding and uptake of the virus. As a result, early membrane-coupled events of SARS-CoV-2 infection are inhibited as mirrored by the decreased binding of spike RBDs to host membrane and decreased cellular uptake of spike trimers, and culminate in decreased cellular infection with replication-competent SARS-CoV-2 virions.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Inhibition, Membrane, Infection, Binding Assay, Clinical Proteomics, Disruption, Virus

Journal: bioRxiv

Article Title: Staged heavy-chain filtering enables Fab discovery from combinatorially intractable library spaces

doi: 10.64898/2026.05.10.724059

Figure Lengend Snippet: (a, b) Binding ELISA curves and apparent half-maximal effective concentration (EC₅₀) values of CLR101 and reference antibodies (CR3022, P2B-2F6, and S309) against the SARS-CoV-2 D614G spike protein (a) and wild-type RBD (b) (n = 2 independent experiments, mean ± s.d.). (c) Apparent EC₅₀ values of CLR101, CR3022, P2B-2F6, and S309 against RBDs of six SARS-CoV-2 variants—wild-type (Wuhan-Hu-1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and BA.5 (B.1.1.529.5)—determined by ELISA (n = 2 independent experiments, mean ± s.d.). (d) Epitope binning analysis of CLR101 by competitive ELISA. The heatmap displays the mean percent inhibition of CLR101 binding to wild-type RBD in the presence of excess competitor proteins (ACE2, CLR101, CR3022, P2B-2F6, and S309). Self-competition by CLR101 was included as a positive control for binding inhibition. The color scale indicates the degree of inhibition from 0% to 100% (n = 3 independent experiments). (e, f) Evaluation of in vitro neutralizing activity against D614G spike-pseudotyped lentiviral particles using hACE2-293T cells. (e) Dose-response neutralization curve of CLR101, showing neutralizing activity with an apparent EC₅₀ of 11.1 ± 2.6 nM (n = 2 independent experiments, mean ± s.d.). (f) Percent neutralizing activity of CLR101 alongside benchmark antibodies at a fixed antibody concentration of 100 nM (n = 2 independent experiments, mean ± s.d.).

Article Snippet: Virus–antibody mixtures were then added to monolayers of hACE2-expressing 293T cells (hACE2-293T; Takara Bio Inc., Kusatsu, Shiga, Japan) in 96-well plates.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Competitive ELISA, Inhibition, Positive Control, In Vitro, Activity Assay, Neutralization