Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies

<div><p>Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.</p></div

Virus neutralization and cotton rat protection studies.

<p>Neutralization curves based upon a plaque-reduction assay performed in Vero cells in the presence of complement using RSV strain A2 (A) or strain B1 (B) when incubated with CB017.5 IgG (green) or CB002.5 IgG (blue). IC₅₀ values were determined by the mean concentration required to inhibit 50% of RSV infection. (C) Neutralization curves, in the absence of complement, of an <i>in vitro</i> RSV cell culture model using human bronchial epithelial cells (HBECs) cultured at an air-liquid interface, colored as in A and B. (D) Cotton rat lung histopathology scores of each treatment group in the treatment arm of the study were evaluated six days after infection. Slides were scored blindly as described in the methods, with lower scores indicating reduced inflammation and pathology. The red line indicates the median of each group. (E) Infectious RSV titers in the lungs of cotton rats four days post-infection as determined by plaque assay. Different animal groups were injected prophylactically 24 hours prior to infection, or as a treatment one day after infection, as indicated. Animals used to determine viral titers were not included in the histopathological analysis. The red line indicates the mean viral titer. Gray dots indicate viral titers that were at the lower limit of detection. For panels (D) and (E): *p <.05, **p <.01, ns = not significant.</p

Crystal structure of Fab CB017.5 bound to an RSV G peptide.

<p>(A) Ribbon diagram of the Fab CB017.5 variable domain (Fv) in complex with the subtype A RSV G peptide. (B) Ribbon-and-stick representation of Fab CB017.5 in complex with the G peptide. Black dotted lines indicate hydrogen bonds and the red dotted line indicates a salt bridge. (C) Ribbon-and-stick representation of the G peptide and a molecular surface representation of Fab CB017.5, rotated 45° from the view in (B). In panels (A–C) the heavy chain is colored green, the light chain is colored white, and the G peptide is colored on a spectrum from yellow to red, N- to C-terminus, respectively. For stick models, oxygen atoms are colored red, nitrogen are blue, and sulfur are yellow.</p

Fabs CB017.5 and CB002.5 bind with high affinity to RSV G and a G peptide.

<p>Surface plasmon resonance (SPR) response curves of Fab CB002.5 (top) and Fab CB017.5 (bottom) binding to wild-type RSV sG from strain A2 (A) and a subtype A RSV G peptide encompassing the central conserved region (B). The raw data are plotted in black, and the calculated best fit to a 1:1 binding model is plotted in red. The equilibrium dissociation constant (<i>K</i>_D) for each interaction is displayed above the respective SPR curve. (C) Sequence alignment of the 45-residue G peptide and the corresponding region of RSV G from strains A2 and B1. The strictly conserved residues, the cystine noose, and CX3C motif are labeled.</p

Structural comparison of the Fab–G peptide complexes.

<p>(A) Ribbon representation of the Fab CB002.5–G peptide (left) and Fab CB017.5–G peptide (right) complexes, generated by superimposing the structurally conserved cystine noose. Each complex is shown separately for clarity and colored as in Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006935#ppat.1006935.g003" target="_blank">3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006935#ppat.1006935.g004" target="_blank">4</a>. (B) Ribbon representation of the G peptide, shown in the conformation adopted when bound to Fab CB002.5 (blue) or Fab CB017.5 (green). The figure was generated by superimposing the cystine nooses of the G peptides (Cys173–Cys186). The two disulfide bonds are shown as sticks, with sulfur atoms colored yellow.</p

Structural comparison of RSV G and fractalkine CX3C motifs.

<p>(A) Ribbon diagram of fractalkine (left, purple, PDB ID: 1F2L) and the RSV G peptide, shown in the conformation adopted when bound to Fab CB002.5 (right, blue). The CX3C motif is displayed as red sticks and the sequence is shown below each structure. (B) Ribbon diagram of fractalkine bound to US28, a viral-homolog of human CX3CR1 (shown as a white molecular surface; PDB ID: 4XT1), rotated 180° from the view of fractalkine in (A). For stick models in panels (A) and (B), oxygen atoms are colored red, nitrogen are blue, and sulfur are yellow.</p

Crystal structure of Fab CB002.5 bound to an RSV G peptide.

<p>(A) Ribbon diagram of the Fab CB002.5 variable domain (Fv) in complex with the subtype A RSV G peptide. (B) Ribbon-and-stick representation of the interaction between the CDR H3 of Fab CB002.5 and the G peptide. Black dotted lines indicate hydrogen bonds and the red dotted line indicates a salt bridge. (C) Ribbon-and-stick representation of the G peptide and a molecular surface representation of Fab CB002.5, rotated 110° from the view in (B). In panels (A–C) the heavy chain is colored blue, the light chain is colored white, and the G peptide is colored on a spectrum from yellow to red, N- to C-terminus, respectively. For stick models, oxygen atoms are colored red, nitrogen are blue, and sulfur are yellow.</p