Fig 1.
AM14 is a prefusion-specific neutralizing antibody.
(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.
Fig 2.
Structures of AM14 in complex with RSV F.
(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).
Table 1.
Amino acid changes in F protein of AM14 MARMs.
Fig 3.
N426D disrupts binding of AM14 to prefusion F.
Relative binding of D25, 101F, MPE8 and AM14 to cell surface-expressed prefusion F (grey) or prefusion F containing the AM14 escape mutation, N426D (white) was measured by flow cytometry. Data were normalized to motavizumab binding. Binding of D25, 101F and MPE8 to N426D was comparable to wild-type prefusion F, whereas AM14 binding to N426D was reduced four-fold (n = 3, P < 0.001; Tukey’s HSD, error bars show standard deviation).
Fig 4.
AM14 is specific for cleaved, trimeric RSV F.
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.
Fig 5.
AM14 stabilizes RSV F trimer in the absence of the foldon trimerization motif.
Size-exclusion chromatography profiles from a Superose 6 column are shown for AM14 Fab or D25 Fab complexed with prefusion RSV F containing the foldon trimerization motif (black and grey, respectively) and for AM14 Fab or D25 Fab co-expressed with RSV F ectodomain without foldon (red and blue, respectively).