Research ArticleINFECTIOUS DISEASE

Structures of respiratory syncytial virus G antigen bound to broadly neutralizing antibodies

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Science Immunology  09 Mar 2018:
Vol. 3, Issue 21, eaar3534
DOI: 10.1126/sciimmunol.aar3534
  • Fig. 1 bnmAbs 3D3 and 2D10 bind RSV G161-197.

    (A) Schematic of the RSV G glycoprotein from RSV strain A2, including the transmembrane region (TM), CCD, and the cysteine noose (Cys noose). Met48 is the alternate initiation site for the production of soluble RSV G. Predicted N- and O-linked glycans are shown by black “N” and gray “O,” respectively. (B) Sequence alignment of RSV G CCD from diverse RSV strains. Amino acids 157 and 198 are predicted to be O-glycosylated in RSV strain A2 (gray O) and represent the boundaries. Secondary structure, disulfide bonds, and heparin-binding domain (HBD) are displayed. Amino acids within bnmAb 3D3 and 2D10 epitopes (antigenic sites γ1 and γ2) are labeled with blue and orange circles, respectively. (C) Coomassie-stained SDS-polyacrylamide gel of RSV Gecto and RSV G161-197. (D) ELISA showing binding of bnmAbs 3D3 and 2D10 to RSV Gecto. (E) ELISA showing binding of bnmAbs 3D3 and 2D10 to RSV G161-197. ELISA experiments were performed in biological triplicates. Error bars indicate SEM.

  • Fig. 2 Crystal structures of RSV G-antibody complexes.

    Overall view (top) and zoom-in view (bottom) of the (A) Fab 3D3-RSV G162-172 complex, (B) Fab 3D3-RSV G161-197 complex, and (C) the scFv 2D10-RSV G169-198 complex. In all panels, RSV G is colored cyan, bnmAb heavy chain is colored dark gray, and bnmAb light chain is colored light gray. Water molecules are shown in red. Hydrogen bonds are shown as dashes. Heavy-chain CDRs (HCDR1-3) and light-chain CDRs (LCDR1-3) are labeled.

  • Fig. 3 bnmAbs 3D3 and 2D10 specifically block RSV G161-197–induced chemotaxis.

    Chemotaxis assays were performed in a Transwell plate, with THP-1 cells added to the upper chamber and chemoattractant added to the lower chamber. (A) Serum-free media were used as negative control, 10% FBS as positive control, 5 nM RSV Gecto, or 5 nM RSV G161-197. (B) Serum-free media containing BSA (1 mg/ml) were used as a negative control and 10 nM CX3CL1 (fractalkine) as a positive control. Chemotactic indices were determined by comparing the fold increase in cell migration toward the chemoattractant compared to cell migration toward serum-free media alone. Studies with bnmAbs (25 nM) were used to examine inhibition of RSV G161-197–induced and CX3CL1-induced chemotaxis. Studies with anti-CX3CR1 preincubated with THP-1 cells in the upper chamber were used to examine antagonism of cell migration toward RSV G161-197 and CX3CL1 in the lower chamber. Student’s t test was performed. Black asterisks denote significance compared to negative control and gray asterisks denote significance compared to RSV G161-197 (A) or CX3CL1 (B); ***P < 0.001, ****P < 0.0001. Chemotaxis experiments were performed in four biological replicates. Error bars indicate SEM.

  • Fig. 4 Sequence conservation within antigenic sites γ1 and γ2, atomic interactions within the RSV G CCD, and model of RSV G glycoprotein.

    (A) Top: Surface representation of RSV G with amino acids colored according to conservation. Atoms from the main chain and conserved side chains are red, similar side chains are pink, and nonconserved side chains are white. The five CX3C motif amino acids are outlined in green. Middle and bottom: The epitope footprints of antigenic site γ1 (bnmAb 3D3 epitope) and antigenic site γ2 (bnmAb 2D10 epitope), respectively. (B) Structure of RSV G161-197 with hydrogen bonds shown in purple dashes and representative side chain–side chain hydrophobic interactions (≤4.0 Å) shown in gray dashes. (C) Schematic of membrane-bound RSV G. N- and O-linked glycans are shown by black and gray discs, respectively.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/21/eaar3534/DC1

    Fig. S1. Co-elution of RSV G-antibody complexes in solution.

    Fig. S2. Electron density maps and structural alignments of RSV G.

    Fig. S3. Schematic of RSV G interactions with anti-G antibodies.

    Fig. S4. Structural comparison of RSV G with CX3CL1 (fractalkine).

    Fig. S5. Isolated human anti-G mAbs and epitope characterization.

    Table S1. Crystallographic data collection and refinement statistics.

  • Supplementary Materials

    Supplementary Material for:

    Structures of respiratory syncytial virus G antigen bound to broadly neutralizing antibodies

    Stanislav O. Fedechkin, Natasha L. George, Jacob T. Wolff, Lawrence M. Kauvar, Rebecca M. DuBois*

    *Corresponding author. Email: rmdubois{at}ucsc.edu

    Published 9 March 2018, Sci. Immunol. 3, eaar3534 (2018)
    DOI: 10.1126/sciimmunol.aar3534

    This PDF file includes:

    • Fig. S1. Co-elution of RSV G-antibody complexes in solution.
    • Fig. S2. Electron density maps and structural alignments of RSV G.
    • Fig. S3. Schematic of RSV G interactions with anti-G antibodies.
    • Fig. S4. Structural comparison of RSV G with CX3CL1 (fractalkine).
    • Fig. S5. Isolated human anti-G mAbs and epitope characterization.
    • Table S1. Crystallographic data collection and refinement statistics.

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