like bounds  (Routledge Ltd)

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A) Neutralization of live EBOV, SUDV, or BDBV by the indicated monoclonal antibodies (mAbs) was assessed by plaque assay. Left: Neutralization of EBOV by varying concentrations of each mAb. Right: Summary table showing the 50 or 80% plaque reduction neutralization test (PRNT50 or PRNT80) for each mAb against the indicated viruses. (B) Grouping of the neutralizing mAbs into competition groups. The numbers in the table represent the binding of fluorescently labeled mAbs (columns) to EBOV GP-expressing cells in the presence of excess unlabeled competitor mAbs (rows). Binding in the presence of each competitor is expressed as a percentage of the fluorescence signal in the absence of competitor. mAbs were classified as soluble glycoprotein (sGP)-binding (+) or non-binding (−) by enzyme-linked immunosorbent assay (ELISA). mAb 9.20.1C3 was unique within the chalice bowl competition group in being unable to bind to sGP (yellow highlight).

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques: Neutralization, Plaque Assay, Plaque Reduction Neutralization Test, Binding Assay, Labeling, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A). Side view. Cryogenic electron microscopy (cryo-EM) map in transparent grey and ribbon model of the complex. A single 1C3 antigen-binding fragment (Fab; orange) binds into the center of the glycoprotein (GP) trimer, with density visible for the entire Fab (grey), with the variable fragment (Fv) modeled in orange. (B) Molecular surface of EBOV GP in grey, with a single bound 1C3 reaching down into the chalice bowl. (C) Top view. Cryo-EM map in transparent grey. Three 1C11 antibodies, Fv modeled in blue, bind to GP, bridging the fusion loop to an adjacent GP protomer. (D) Molecular surface with the single 1C3 Fab (orange ribbon) extending diagonally across the entire GP trimer.

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques: Electron Microscopy, Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A–C) or surface representation (D, F). 1C11 single-chain variable fragment (scFv) is shown in blue, and 1C3 antigen-binding fragment (Fab) is shown in orange. The international immunogenetics information system (IMGT) numbering scheme is used for mAbs 1C11 and 1C3. (A) Closeup of 1C11-fusion loop interaction with key side chains of 1C11 (blue) and GP (grey) illustrated. GP residues that are conserved among five ebolaviruses (EBOV, SUDV, BDBV, RAFV, and RESTV) are underlined. (B) 1C11 draws the fusion loop away from the core of GP upon binding. The conformation of the fusion loop in an unbound GP (PDB:5JQ3) is shown in green, and the 1C11-bound conformation is shown in black. (C) The N-linked glycan at position 563 and the 1C11 complementarity determining region (CDR) side chains that interact with it are illustrated. Side chains within hydrogen bonding range to the glycan are indicated by dashed yellow lines. (D) 1C3 and the head region of EBOV GP are shown in closeup, including the long CDRL1 loop of 1C3 which extends deep into the EBOV GP “chalice”. (E) The footprints of 1C3 and 1C11 are highlighted on the sequence alignment of five major ebolaviruses. Residues were labeled by colored triangles at the bottom of the alignment to show the conservation (blue - conserved, pink - similar, yellow - non-conserved). The footprint of 1C3 contains five non-conserved residues, and the footprint of 1C11 contains only conserved or highly similar residues. (F) On the left, EBOV GP surfaces that directly contact 1C3 are shown in various shades of orange as follows: 1C3-bound residues in common with all three GP protomers are colored in dark orange, residues bound by only two protomers (A and B) are colored in medium orange, and residues unique to a single protomer are colored in light orange and yellow. On the right, the three separate portions of the tripartite 1C3 footprint on the GP protomers A, B, and C are illustrated. For example, residues 114–120 on protomer A are bound by CDRs H2 and H3, while the same residues 114–210 on protomer B are bound by 1C3 CDRL2 and framework region 3. CDRL1 of 1C3 simultaneously contacts residues 124–126 on both protomers A and B.

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques: Binding Assay, Sequencing, Labeling

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A) Amino-acid residue changes in 1C3. Only one change in 1C3, W111A, compromised tight binding of Ebola virus (EBOV) glycoprotein (GP). All other single amino-acid residue changes could be accommodated, presumably by other key contacts remaining. (B and C) Changes in GP residues. (B) Point changes in GP at 3 of 4 key residues result in reduced binding by 1C3, presumably because these single amino acids form more than one contact point each in the tripartite epitope. (C) Point changes at all four key residues result in reduced neutralization of recombinant vesicular stomatitis virus expressing EBOV GP (rVSV-EBOV) by 1C3. (D) Viral escape mutants from 1C3 and 1C11. Left column: Mutations identified in plaque-purified ΔVP30 Ebola virus isolates grown in the presence of 1C3 or 1C11. Right column: Mutations identified in Jurkat cell lines expressing randomly mutated EBOV GP that were selected for loss of binding to 1C3 or 1C11 by FACS sorting.

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques: Binding Assay, Neutralization, Recombinant, Expressing, Purification

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A) Survival of mice treated with 1C3 or 1C11 prior to Ebola virus (EBOV) exposure. mAbs were administered 24 h prior to exposure with 100 plaque-forming units (PFU) of mouse-adapted EBOV. A human immunoglobulin G against influenza A virus (IgG1) mAb was used as a control. Animal survival was assessed twice daily for 21 d. n = 10 mice were studied per treatment condition. (B) Survival of STAT1 KO mice treated with 1C3 or 1C11 after EBOV/BDBV-GP exposure. Groups of STAT1 KO mice at five animals per group were injected with the indicated mAbs by the intraperitoneal route at 24 h after BDBV chimeric virus challenge. Kaplan-Meier survival curve is shown. Each group was compared with phosphate-buffered saline (PBS) control (Mantel-Cox test). (C and D) Groups of guinea pigs at five animals per group were injected with indicated mAbs by the intraperitoneal route at 1 or 3 days after EBOV (C) or Sudan virus (SUDV) (D) exposure. Kaplan-Meier survival curves, body weights, illness score curves, and viremia levels are shown. For analysis of survival, each group was compared to PBS mock control (Mantel-Cox test). In panels representing viremia, each dot corresponds to an individual serum sample. Short horizontal lines indicate the mean value of titers. The dotted horizontal lines show the detection limit. For analysis of viremia data, serum samples collected on different days were pooled together in each experimental group. Samples without detectable virus were arbitrarily assigned the viremia level values corresponding to the detection limit (102 PFU/mL). One-way analysis of variance (ANOVA) with Dunnett correction was used for multiple comparisons between each group and PBS mock control.

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques: Injection

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A) Complete survival of crab-eating macaques (n=3) exposed to Sudan virus (SUDV) following treatment with 50 mg/kg of 1C3+1C11 on Day 4 or Day 7 post-exposure, as compared to mock-treated control nonhuman primates (NHPs; n=2). (C) Complete survival of rhesus monkeys treated with 50 mg/kg or 25 mg/kg of 1C3+1C11 on Day 4 or Day 7 following EBOV exposure as compared to a mock-treated NHP (n=1) (C). (B and D) Infectious titers (log10 per mL serum) and viral genome equivalents (GE) per mL serum are shown over time for each animal following SUDV (C) and EBOV (D) exposure.

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques:

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: Key resources table

Article Snippet: Structure of EBOV GP lacking the mucin-like domain with 1C11 scFv and 1C3 Fab bound (cryo-EM) , This study , EMD-25471.

Techniques: Labeling, Luciferase, Recombinant, Blocking Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Modification, TaqMan Assay, Plasmid Preparation, Software, High Content Screening, Microscopy, Real-time Polymerase Chain Reaction