Structural Basis for Postfusion-Specific Binding to the Respiratory Syncytial Virus F Protein by the Canonical Antigenic Site I Antibody 131-2a

Abstract

The respiratory syncytial virus (RSV) fusion (F) protein is a major target of antiviral antibodies following natural infection or vaccination and is responsible for mediating fusion between the viral envelope and the host membrane. The fusion process is driven by a large-scale conformational change in F, switching irreversibly from the metastable prefusion state to the stable postfusion conformation. Previous research has identified six distinct antigenic sites in RSV-F, termed sites Ø, I, II, III, IV, and V. Of these, only antigenic site I is fully specific to the postfusion conformation of F. A monoclonal antibody 131-2a that specifically targets postfusion F has been widely used as a research tool to probe for postfusion F and to define antigenic site I in serological studies, yet its sequence and precise epitope have remained unknown. Here, we use mass spectrometry-based de novo sequencing of 131-2a to reverse engineer a recombinant product and study the epitope to define antigenic site I with molecular detail, revealing the structural basis for the antibody's specificity toward postfusion RSV-F.

Keywords: 131−2a; antibody sequencing; antigenic site I; epitope mapping; fusion protein; respiratory syncytial virus.

1

Antigenic sites on the RSV-F protein. The mature RSV-F antigen is proteolytically processed from the F0 precursor into disulfide-linked F1 and F2 subunits, releasing the p27 peptide. FP: fusion peptide, TM: transmembrane region. (left) Prefusion conformation of RSV-F (PDB ID: 4MMU). (right) Postfusion conformation of RSV-F (PDB ID: 3RRR). Antigenic sites are plotted on the surface with the indicated colors. Specificity of the antigenic sites for pre- and postF is indicated with the colored dots. Antigenic site definition follows Hause et al. 2017. The monoclonal antibody 131–2a is widely used to define antigenic site I.

2

De novo sequencing of 131–2a by mass spectrometry-based bottom-up proteomics. The variable region alignment to the inferred germline sequence is shown for both heavy and light chains. Positions with putative somatic hypermutation are highlighted with asterisks (*). The MS2 spectra supporting the complementarity determining regions (CDRs) are shown beneath the sequence alignment.

3

Validation of recombinant 131–2a expressed with a mass spectrometry-derived sequence. (A) ELISA of pre/postF comparing the sequenced input and reverse-engineered 131–2a. (B) Western blot analysis following nondenaturing PAGE of pre/postF comparing the sequenced input and reverse-engineered 131–2a. The bands at 100 kDa and >250 kDa represent F monomers and trimers, respectively.

4

Atomic model of RSV postF bound to the 131–2a Fab. (A) Surface representation of postF bound to a single copy of 131–2a’s VH/VL domains. (B) Detailed view of the buried surface between 131–2a (paratope) and postF (epitope).

5

Detailed interactions in the 131–2a epitope on postF. (A) Interactions with residues 379–399 of the F1 subunit. (B) Interactions with residues 323–332 (C322–C333 loop) in the F1 subunit. (C) Interactions with residues 31–42 of the F2 subunit. Key residue numbers with associated chains are indicated, and hydrogen bonds are indicated with dashed blue lines. The RSV-F sequence is highly conserved within subgroups A and B, with the corresponding consensus sequences of the epitope displayed. Hydrogen-bonded residues to 131–2a are underlined.

6

Structural basis for postF specificity of 131–2a. (A) Surface representation of the postF crystal structure (PDB ID: 3RRR). (B) Surface representation of the preF crystal structure (PDB ID: 4MMU). (C) Cartoon representation of a single preF protomer at the 131–2a epitope. While the conformation of the 131–2a epitope remains the same in preF versus postF, access to the epitope is blocked in preF by the α10 helix and heptad repeat B (HRB) of the F1 C-terminus, which refolds into the helical stalk region of postF.

7

Comparison of the 131–2a epitope (A) with the antigenic site I antibody ADI–14359 (PDB ID: 6APB) (B). The cartoon in (C) shows the ADI–14359 epitope mapped onto a single protomer in preF (PDB ID: 4MMU). While the ADI–14359 epitope remains exposed, the extended C-terminus of F1 augments the β-sheet of the F2 region, sterically clashing with the light chain of ADI–14359 (D).