Fig 1.
RSB1 neutralization and competition with other monoclonal antibodies.
A) Neutralization of RSV A and B strains in a plaque reduction assay by RSB1, D25 and Motavizumab (Mota). IC60 neutralization titer is shown with bars colored blue for RSV strain A, and red for strain B. Error bars represent standard error based on triplicate experiments. B) Biolayer interferometry competition assay. The table is colored based on the percentage of competition of the primary mAbs over the secondary mAbs: green 0–29%, light green 30–49%, light orange 50–69% and red 70–100%. Numbers in parenthesis represent standard deviation from three independent experiments.
Fig 2.
X-ray structure of the DS-Cav1-RSB1 complex.
A) The structure of the DS-Cav1-RSB1 complex is depicted with surfaces colored in light and dark gray for two DS-Cav1 protomers, and orange and gold for the F2 and F1 subunits of the third protomer. Fab RSB1 is depicted with pink and magenta surfaces for the L and H chains, respectively. B) Zoomed view of the region boxed in A), after a rotation of ~90 degrees around the y axis. RSB1 CDR loops are depicted as cartoons and labelled. The total RSB1 epitope surface is colored cyan. C) Same view as in B) with the epitope hydrogen bonding residues on DS-Cav1 colored purple and the residues making van der Waals contacts colored green. RSB1 CDR residues making contacts with the purple and green regions are shown as sticks. D) Zoomed view of the RSB1 epitope on DS-Cav1, to highlight salt bridge interactions (sticks and dashes) only. For clarity, this view is slightly re-oriented with respect to panels B and C. E) Rotated and zoomed view of panel C highlighting RSB1 cross-protomer interactions with DS-Cav1 (dashes).
Fig 3.
Experimental RSB1 binding affinities to DS-Cav1 and its mutants.
Surface Plasmon Resonance (SPR) experiments to analyze binding affinity of RSB1, D25, and AM14 antibodies upon mutation of identified critical residues shown as fold change, with corresponding KD graph shown as an inset. There is a decrease in RSB1 binding due to the single point mutation K65A and the double point mutation N63A/K65A. No change in affinity is observed with the introduction of the RSV B substitutions D200N or N276S.
Fig 4.
Structural comparison between DS-Cav1-RSB1 and DS-Cav1-D25 complexes.
A) RSB1 is depicted as dark and light pink surfaces, D25 in blue and cyan. B) Rotated view of A. C) Zoomed view of boxed region in B reveals the location of residues Asn63 and Lys65, depicted as sticks.
Fig 5.
RSB1 and CR9501 target similar epitopes with different angles of approach and an “induced fit”.
Three views, each rotated by 45 degrees for A) DS-Cav1-RSB1 complex and B) CR9501 complex. For simplicity, only one protomer is shown bound by one Fab. CR9501 is colored dark green for the heavy chain and light green for the light chain. RSB1 coloring matches Fig 1. C-D) Open-book views of the RSB1 (C) and CR9501 (D) interfaces showing total buried surface areas for their respective epitopes and paratopes. E-F) HCDR3 for RSB1 (E) and CR9501 (F) are shown as cartoons, while F1 and F2 are depicted as surface. Residues Leu100 (RSB1) and Ile100A (CR9501) are shown with sticks. Tyr98 on CR9501 which causes an induced fit is also shown as sticks.
Fig 6.
Structural comparison between DS-Cav1-RSB1 and CR9501 complexes reveals an induced fit and convergent structural features.
A-B) View down the membrane distal apex of the prefusion F molecule for RSB1 (A) and CR9501(B) complexes. The α1 helix is shown in cartoon and colored orange. Protomer 2 is colored white. Residues on PreF that shift upon binding of CR9501, due to an “induced fit” caused by CR9501 Tyr98 are shown as sticks. The black arrow shows where RSB1 forms cross protomer interactions that are not found in the CR9501 complex. C) Superposition of the RSB1 and CR9501 complexes showing a zoomed-in view of the “induced fit”. For clarity, the helix for the CR9501 bound complex is shown as transparent cartoon. Residues shifted when CR9501 binds are shown as sticks with a green transparent surface, whereas the corresponding residues on the RSB1 complex are shown as sticks with no surface. The HCDR3 is shown as cartoon for each Fab, with the CR9501 Tyr98 shown as a stick. (D-E) Zoomed view of the LCDR1 Tyr32 interaction with F1 for RSB1 (D) and CR9501 (E).
Fig 7.
Computational analysis of RSB1 binding.
A) Calibration of Rosetta in silico computations by comparison of experimental data with in silico binding affinity calculations for RSB1 and D25 alanine mutations, and RSB1 RSV B substitutions. Mutation free energy is calculated as the change in Gibbs Free Energy (ΔΔG) between the mutant and wildtype residues. B) Difference in pairwise free energy (kcal/mol) for an in silico Tyr32Ala mutation in the RSB1 and CR9501 LCDR1. PCC: Pearson’s Correlation Coefficient RMSE: Root Mean Square Error.
Fig 8.
RSB1-like antibodies are induced upon immunization with DS-Cav1.
A) In a naturally-primed bovine model, RSB1-competing antibodies trend similar to D25-competing antibodies. RSV A neutralization titers are represented by the broken black line. The PostF antigen did not induce measurable neutralizing, or RSB1- or D25-competing antibodies in this study. *The values for D25-like antibodies and RSV A neutralization titers were previously reported in Steff et al., Nature Communications (2017) [17].