Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) On top, cartoon diagram of the SARS-CoV-2 spike with highlighted coldspot areas at the fusion peptide (FP, red), heptad repeat 2 region (HR2, blue), and subdomain 1 (SD1, green). Thick horizontal lines indicate the location of all coldspots (see also fig. S1A). At the bottom, amino acid changes in SARS-CoV-2 variants. Each circle represents a single aa substitution over ancestral virus. (B) Structure of the SARS-CoV-2 spike; FP (aa 814–838) and HR2 (aa 1142–1161) coldspots are in red and blue, respectively (PDB: 6XM4). (C) ELISA measurements of convalescent plasma IgG reactivity to FP (top) or HR2 (bottom) peptides. Optical density units at 450 nm (OD, Y axis) and reciprocal plasma dilutions (X axis). Non-infected controls in black; samples selected for cell sorting by flow cytometry are in red or blue. Two independent experiments. (D) Representative flow cytometry plots of B cells binding to fluorescently labeled FP (top) or HR2 (bottom) peptides. Numbers indicate percentage of double-positive cells in the gate. (E) Number of heavy and light chain V gene somatic mutations of antibodies to the FP (top) or HR2 (bottom) peptides. (F) Heatmaps with ELISA EC 50 values of monoclonal antibodies binding to the S of CoVs (top) and to the FP and HR2 peptides (bottom) corresponding to the CoV species, whose genus is indicated by Greek letters. The monoclonal antibodies to the HR2 region S2P6 and CV3–25 were assayed alongside for comparison. Cross indicates not tested. Two experiments. (G) Graph with IC 50 values of monoclonal antibodies neutralizing pseudoviruses corresponding to the indicated VOC. Two experiments. (H) ACE2 binding to ancestral S in ELISA in the presence of select FP and HR2 antibodies. Dotted line represents the limit of detection. Two experiments. (I) Inhibition of cell fusion by FP and HR2 antibodies. (J and K) fp.006 and hr2.016 antibodies protect in vivo. Top, diagram of the experiment’s timeline. Middle, mouse weight over time after challenge with ancestral SARS-CoV-2 of AAV-hACE2 mice treated with antibodies either 24 hours before ( (J) ; n = 6 per group, p = 0.0022 for both fp.006 and hr2.016 versus isotype at day 7), or 2 hours after ( (K) ; n = 5 per group, p = 0.0079 for both fp.006 and hr2.016 versus isotype at day 7) the infection. Mann-Whitney U test, standard deviation is shown. At the bottom, representative lung images at day 7.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, FACS, Flow Cytometry, Binding Assay, Labeling, Inhibition, In Vivo, MANN-WHITNEY, Standard Deviation

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) Overview of the complex structure of fp.006 Fab (surface representation; heavy chain in teal, light chain in light teal) bound to the SARS-CoV-2 FP (orange cartoon) with interacting side chains represented as sticks. (B) Visualization of FP residues F 823 , E 819 , and R 815 resting in a deep groove formed at the antibody paratope, with coloring as in ( A ). (C) Overlay of the fp.006-FP crystal structure with a cryo-EM structure of the SARS-CoV-2 prefusion S trimer (PDB: 6VXX). Models were aligned on Cα atoms of FP residues 818–822 (helical in both structures) with a root mean square deviation of 0.97 Å. (D) Residue-level interactions between FP residue R 815 and the antibody heavy chain include hydrogen bond formation with N 31 and a cation-π interaction with Y 52A . (E) Water-mediated interactions between FP residue E 819 and heavy chain residues Y 52A , N 56 , and F 97 . Water molecules are shown as red spheres. (F) van der Waals contacts between FP residue F 823 (orange stick) and residues that comprise a groove at the heavy and light chain interface (teal surfaces). (G) Interactions between FP residue D 820 and fp.006 CDRH2 residues include a salt bridge with R 55 and additional hydrogen bond formation with N 56 . Hydrogen bonds, salt bridges, and cation-π interactions are shown as dashed blue lines. (H) Flow cytometry detection of anti-FP and anti-HR2 antibody binding to SARS-CoV-2 S expressed on 293 T cells. Left, representative FACS plots (pre-gated on live-singlets-GFP + cells). Black lines indicate isotype control in the presence (continuous line) or absence (dotted line) of soluble ACE2. Right, quantification of the geometric mean fluorescent intensity (gMFI; n = 3). Two-tailed paired t-test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; standard deviation is shown.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Cryo-EM Sample Prep, Flow Cytometry, Binding Assay, Two Tailed Test, Standard Deviation

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) Structure of the SARS-CoV-2 S. S protomer with RBD up (left) or down (middle) and S trimer with two down and one up (right; PDB: 6XM4). SD1 and RBD are in green and yellow, respectively. (B) Graph shows ELISAs measuring plasma IgG reactivity to SD1-RBD. Negative controls in black; samples selected for sorting in green. Mean of two independent experiments. 82.1% of the plasma samples were positive (4SD higher than the average AUC of the controls) (C) Representative flow cytometry plot of B cells binding to fluorescently labeled SD1-RBD. Percentage refers to gated cells. (D and E) ELISAs measuring the reactivity of monoclonal antibodies to SD1-RBD ( D ) and to RBD ( E ). Mean of two independent experiments. (F) Heatmaps with the binding (EC 50 ) of SD1 monoclonal antibodies to S (top) or SD1-RBD (bottom) proteins corresponding to SARS-CoV-2 VOC. Two experiments. (G) Graph shows normalized relative luminescence values in cell lysates of 293T ACE2 cells after infection with ancestral SARS-CoV-2 pseudovirus in the presence of increasing concentrations of broadly cross-reactive SD1 monoclonal antibodies. At least two independent experiments.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Flow Cytometry, Binding Assay, Labeling, Infection

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) ACE2 binding to ancestral S in ELISA in the presence of sd1.040 or C121 control antibody. Representative of two experiments. (B) Structure of the sd1.040-S complex. Spike SD1 and RBD regions are shown as surface representation and colored wheat and gray, respectively. The sd1.040 Fab heavy chain (dark green) and light chain (light green) are shown as cartoon. The S N331-glycan that interacts with the sd1.040 Fab is shown as teal spheres. Inset: sd1.040 binding orientation on trimeric S shows clashes. (C and D) Surface rendering of sd1.040 epitope is highlighted on the SD1 and RBD surfaces, with sd1.040 CDR loops shown (ribbon). The majority of sd1.040 contacts are mediated by CDRH2, CDRL1 and CDRL3 loops. (E) Surface plasmon resonance (SPR) experiment showing the binding of sd1.040 Fab to ancestral SD1-RBD or S. (F) Antibody sd1.040 prevents ACE2-induced rearrangements. Flow cytometry detection of fp.006 binding to ancestral SARS-CoV-2 S expressed on 293 T cells. Left, representative FACS plots. Black lines indicate isotype control in the presence (continuous line) or absence (dotted line) of soluble ACE2. Right, quantification of the geometric mean fluorescent intensity (gMFI; n = 4). Two-tailed unpaired t-test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Flow Cytometry, Two Tailed Test

Journal: Science Immunology

Article Title: Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein

doi: 10.1126/sciimmunol.ade0958

Figure Lengend Snippet: (A) SPR assay of the sequential binding of immobilized antibodies to SD1-RBD protein followed by either sd1.040, rbd.042 or CoV-X4042. (B) SPR analysis showing that both arms of CoV-X4042 bind simultaneously to the same SD1-RBD molecule, since avidity is retained at decreasing SD1-RBD concentrations. Increasing normalized kd values indicate loss of avidity. Solid lines, IgG; dotted lines, Fab (see also fig. S8A). (C) Normalized relative luminescence values in cell lysates of 293T ACE2 cells after infection with ancestral SARS-CoV-2 pseudovirus in the presence of increasing concentrations of CoV-X4042 or its parental monoclonal antibodies individually or as a cocktail. Isotype control in black. On the right: mean IC 50 values and significance (P) when parental antibodies are compared to CoV-X4042 (n = 4; Welch’s t-test, two-tailed). (D) Graph with IC 50 values of bispecific and parental monoclonal antibodies neutralizing pseudoviruses corresponding to the indicated VOC. Mean of two independent experiments. (E) In vitro neutralization of SARS-CoV-2 by CoV-X4042. (F) CoV-X4042 protects in vivo. Top, diagram of the experiment’s timeline. Bottom, mouse weight over time after challenge, with ancestral SARS-CoV-2, of AAV-hACE2 mice treated with antibodies either 24 hours before (PRE; n = 5 per group, p = 0.0079), or 2 hours after (POST; n = 5 per group, p = 0.0079; day 7) the infection. Mann-Whitney U test, standard deviation is shown. (G) CoV-X4042 reduces viral titers in the lungs. Mice were treated with antibodies 24 hours before infection and virus titers evaluated on day 3 (n = 5 per group p = 0.0079 with both ancestral and Omicron BA.1; Mann-Whitney U test, standard deviation is shown).

Article Snippet: 293T ACE2/TMPRSS2 cell line was generated by transfecting 293T ACE2 ( ) cells with pCMV3-FLAG-TMPRSS2 (SinoBiological) using Lipofectamine 3000 (Invitrogen) and selected with 200 μg/mL Hygromycin B (Invivogen) two days post-transfection.

Techniques: SPR Assay, Binding Assay, Infection, Two Tailed Test, In Vitro, Neutralization, In Vivo, MANN-WHITNEY, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Single neurons on microelectrode array chip: manipulation and analyses

doi: 10.3389/fbioe.2023.1258626

Figure Lengend Snippet: Cell manipulation on the microelectrode array (MEA) chip: (A) Cells were trapped by varying the diameters of microwells from 5 to 20 µm on one chip. Most of the microwells on the electrodes trapped cells easily; however, only 5-µm-wells trapped single cells rather than multiple cells; (B) 293T cells were trapped on all the electrodes ( left ) and labelled using fluorescence to visualize the captured cells ( right ); (C) A cell was precisely controlled to move freely between electrodes. A single cell was randomly trapped in a single electrode and released by reducing the voltage applied to that electrode. The cell moved with the flow and was trapped by the subsequent electrode. (Scale bar = 50 µm).

Article Snippet: The 293T cell line presented in this study was obtained commercially from Sino Biological Inc. (Beijing, China).

Techniques: Microelectrode Array, Fluorescence

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Single neurons on microelectrode array chip: manipulation and analyses

doi: 10.3389/fbioe.2023.1258626

Figure Lengend Snippet: Neural cells culture, validation, and stimulation on chip: (A) The process of differentiation from induced pluripotent stem cells (iPSCs) to neural progenitor cells (NPCs) and subsequently to neuron cells (NCs). Each step requires approximately 3 weeks to grow (Scale bar = 50 µm); (B) The immunofluorescence staining of neurons. Visualizing the morphology of neurite outgrowth through the detection of microtubule associated protein 2 (MAP2) in red, class III beta-tubulin (TuJ1) in green, and 4′,6-diamidino-2-phenylindole (DAPI) in blue, confirmed the maturation of neurons (Scale bar = 50 µm); (C) 293T cells and neurons were stimulated under different voltages for 5 min. Cell survival rates were evaluated after 24 h of culturing; (D) Neurons were stimulated at 0.6 Vpp for 5 min. The survival rates of neurons in three distinct areas were assessed on days 2, 4, and 6, with a significant decrease observed in the near electric field (NEF) area on day 6; (E) The morphology of neurons in (D) on day 6. Neurons in the NEF area developed multiple symptoms, such as swelling, severe cytoplasmic vacuolization, and plasma membrane blebbing, indicating oncosis, a specific mode of cell death. The neurites of neurons in the electric field (EF) area were thinner and shorter, likely due to the damage caused by the electric field. The neurons in far electric field (FEF) area were healthy, almost unaffected by the electric field. (Scale bar = 100 µm).

Article Snippet: The 293T cell line presented in this study was obtained commercially from Sino Biological Inc. (Beijing, China).

Techniques: Immunofluorescence, Staining, Membrane