Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

<div><p>Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.</p></div

Structures of AM14 in complex with RSV F.

<p>(A) Crystal structure of three AM14 Fabs bound to the prefusion RSV F trimer, viewed from the side and the top of RSV F. Negatively stained EM class averages that may correspond to each view are shown in the upper right. AM14 heavy chain is blue, light chain is white, and RSV F protomers are tan, light green and pink (B) Close-up of the side view, colored as in (A). Prefusion RSV F residues with Cα atoms within 8 Å of AM14 Fab Cα atoms are colored red, and Cα atoms of resistance mutations are shown as spheres. (C) Ninety-degree rotation of the view in (B), showing a molecular surface representation of RSV F and the location of the three AM14 CDR loops that contact F. The binding surface spans the two neighboring protomers (tan and pink).</p

AM14 is a prefusion-specific neutralizing antibody.

<p>(A) Neutralization of laboratory strains and clinical isolates of RSV. Red bars are geometric means. (B) Binding of AM14 and motavizumab IgGs to immobilized RSV F proteins was measured using a Luminex system. (C) Binding of AM14 Fab to immobilized prefusion RSV F was measured by surface plasmon resonance. Best fit of the data to a 1:1 binding model is shown in red.</p

AM14 is specific for cleaved, trimeric RSV F.

<p>Binding of antibodies (A) AM14, (B) D25, (C) MPE8, or (D) palivizumab to uncleaved monomeric RSV F (open black triangles), cleaved monomeric RSV F (black circles), uncleaved postfusion RSV F (open blue triangles), cleaved postfusion RSV F (blue circles), uncleaved prefusion RSV F (open red triangles) and cleaved prefusion RSV F (red circles) was measured by ELISA.</p

Amino acid changes in F protein of AM14 MARMs.

<p>Amino acid changes in F protein of AM14 MARMs.</p