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. 2013 May 31;340(6136):1113-7.
doi: 10.1126/science.1234914. Epub 2013 Apr 25.

Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody

Jason S McLellan  1 , Man Chen, Sherman Leung, Kevin W Graepel, Xiulian Du, Yongping Yang, Tongqing Zhou, Ulrich Baxa, Etsuko Yasuda, Tim Beaumont, Azad Kumar, Kayvon Modjarrad, Zizheng Zheng, Min Zhao, Ningshao Xia, Peter D Kwong, Barney S Graham
Affiliations

Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody

Jason S McLellan et al. Science. .

Abstract

The prefusion state of respiratory syncytial virus (RSV) fusion (F) glycoprotein is the target of most RSV-neutralizing activity in human sera, but its metastability has hindered characterization. To overcome this obstacle, we identified prefusion-specific antibodies that were substantially more potent than the prophylactic antibody palivizumab. The cocrystal structure for one of these antibodies, D25, in complex with the F glycoprotein revealed D25 to lock F in its prefusion state by binding to a quaternary epitope at the trimer apex. Electron microscopy showed that two other antibodies, AM22 and 5C4, also bound to the newly identified site of vulnerability, which we named antigenic site Ø. These studies should enable design of improved vaccine antigens and define new targets for passive prevention of RSV-induced disease.

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Figures

Figure 1
Figure 1. RSV neutralization, F glycoprotein recognition, and crystal structure of human antibody D25 in complex with the prefusion RSV F trimer
The prefusion conformation of RSV F is metastable, and when expressed in a soluble form readily adopts the postfusion state; a number of potent antibodies, including D25, bind to a newly revealed antigenic site at the top of the prefusion F glycoprotein. (A) RSV neutralization by antibodies. Palivizumab is the FDA-approved prophylactic antibody that prevents severe RSV disease. (B) ELISA measuring antibody binding to postfusion F glycoprotein. For (A) and (B), data are representative of multiple independent experiments. (C) D25-RSV F trimer crystal structure in ribbon and molecular surface representations. One protomer of the F glycoprotein trimer is shown as ribbons and colored as a rainbow from blue to red, N-terminus of F2 to C-terminus of F1, respectively. Molecular surfaces are shown for the other two F protomers, colored pink and green. The D25 Fab bound to the F protomer shown in ribbons is also displayed in ribbon representation, with heavy chain colored red and light chain colored grey. The other D25 Fabs are colored the same, but shown in surface representation.
Figure 2
Figure 2. Structural rearrangement of RSV F
To mediate virus-cell entry, the RSV F glycoprotein transitions from a metastable prefusion conformation to a stable postfusion conformation. Outer images display prefusion (left) and postfusion (right) trimeric structures, colored the same as in Fig. 1C. A complex glycan, shown as sticks, is modeled at each of the three N-linked glycosylation sites found in the mature protein. Inner images display a single RSV F protomer in ribbon representation, colored as a rainbow from blue to red, N-terminus of F2 to C-terminus of F1, respectively. Select secondary structure elements are labeled (correspondence with amino acid sequence in pre- and postfusion conformations is shown in fig. S3). Inset: schematic of the mature RSV F protein in the RSV F(+) Fd construct. The rainbow coloring of the boxes representing the F2 and F1 subunits matches that in the structures. Glycans are shown as branches on top of the boxes, and disulfide bonds are shown as black lines under the boxes. Amino acids that move more than 5 Å in the pre- and post-fusion conformations are indicated by black bars.
Figure 3
Figure 3. RSV F interface with D25
Antibody D25 binds a quaternary epitope spanning two protomers at the apex of the prefusion F trimer. (A) Close-up of the interface between D25 and RSV F. Side chains of F residues interacting with D25 are labeled and shown as sticks. Oxygen atoms are colored red and nitrogen atoms are colored blue. Hydrogen bonds are depicted as dotted lines. The two images are related by a 90° rotation about the vertical axis. (B) Position and conformation of the D25 epitope on the prefusion and postfusion F molecules. RSV F residues at the D25 interface are colored red; polarity of α4 and α5post indicated with arrows, with fragment N- and C-termini indicated. (C) Surface representation of RSV F colored according to sequence conservation. Coloring was generated using the ConSurf server (40) using 178 F glycoprotein sequences from human RSV subtype A and B, as well as bovine RSV. (D) Sequence conservation of F residues in regions recognized by D25. Amino acids in human RSV subtype B (hRSV/B) or in bovine RSV (bRSV) that differ from hRSV/A are colored red. Ectodomain is defined as F residues 26-109 and 137-524.
Figure 4
Figure 4. Antigenic site Ø
Highly effective RSV-neutralizing antibodies target a site at the membrane-distal apex of the prefusion F trimer. (A) The ability of antibodies to block D25 binding to RSV-infected cells was measured as a function of antibody concentration. (B) Analysis of RSV F-Fab complexes by negative stain electron microscopy: Reprojection of a 12 Å slice through the crystal structure of RSV F + D25 Fab filtered to 10 Å resolution (left). A slice was used to emphasize visibility of the F glycoprotein cavity. Aligned average of 263 particles of RSV F + D25 Fab (middle - left). Aligned average of 550 particles of RSV F + AM22 Fab (middle - right). Aligned average of 171 particles of RSV F + 5C4 Fab (right). Scale bar is 50 Å. (C) Fusion inhibition and (D) attachment inhibition activity for antibodies targeting antigenic site Ø and F-specific antibodies targeting other antigenic sites. For the attachment-inhibition assay, heparin was used as a positive control. Data in panels (A) (C) and (D) are representative of multiple independent experiments.
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