Mutational constraints on RSV F and its neutralization by antibodies

Figure 1.. RSV F mutations cause more nirsevimab escape in subtype B versus subtype A because the ability of bivalent IgG binding to buffer mutations depends on Fab affinity. (A) Bivalent IgG binding buffers the effects of mutations that reduce monovalent Fab affinity when the Fab has sufficiently high affinity for the viral antigen, but how this buffering vanishes if the Fab affinity is more modest. (B) Predictions of a quantitative biophysical model (see Supplementary Appendix) of how a viral mutation that reduces Fab affinity affects both IgG and Fab neutralization of a viral strain to which the Fab has high or low affinity. When the Fab has high affinity, mutations that reduce affinity do not appreciably reduce IgG neutralization. (C) Experimental data showing how two known RSV subtype B nirsevimab escape mutations, K68Q and (K/N)201S, affect neutralization by nirsevimab IgG and Fab of pseudoviruses expressing RSV F from subtype A (Long strain) or subtype B (B1 strain). Nirsevimab has a higher affinity for subtype A than subtype B33,50. Consistent with the biophysical model, the mutations reduce Fab neutralization for both subtypes, but only reduce IgG neutralization for subtype B.…

Figure 2.. Effects of mutations to RSV F on its cell entry function (A) Effects of single amino acid mutations on pseudovirus entry in 293T-TIM1 cells as measured by deep mutational scanning. Each box represents the effect of a specific mutation, with colors indicating decreased (red), unchanged (white), or slightly increased (blue) cell entry relative to the unmutated F. Mutations not reliably measured in our experiments are indicated by gray boxes, and the wildtype amino acid in the parental subtype A Long strain at each site is indicated with an ‘x’. See https://dms-vep.org/RSV_Long_F_DMS/cell_entry.html for an interactive version of this heatmap. (B) Correlation between mutation effects on cell entry measured by deep mutational scanning and pseudovirus titer in a validation assay using F single-mutant pseudoviruses. Each point represents the mean of two replicate measurements. (C) Average effect of mutations at each site mapped onto the prefusion F trimer structure (PDB 5UDC21), viewed from the top and side. Each protomer is colored with a separate color with darker shading indicating more deleterious effects on cell entry. The nirsevimab and clesrovimab binding footprints are outlined…

Figure 3.. Effects of mutations to RSV F on neutralization by nirsevimab IgG and Fab (A) Total decrease in neutralization from all mutations at each site for nirsevimab IgG or Fab as measured by deep mutational scanning. See https://dms-vep.org/RSV_Long_F_DMS/nirsevimab_neutralization.html for interactive plots that show the effects of all mutations. (B) Heatmaps showing effects on neutralization of mutations within the epitope of nirsevimab. Each box represents the effect of a single mutation, with positive values indicating decreased neutralization. Dark gray indicates mutations that are too deleterious for cell entry to measure their effect on neutralization. The wildtype amino acid in the subtype A Long strain used for the deep mutational scanning at each site is indicated with an ‘x’. (C) Pre-fusion RSV F trimer (PDB 5UDC21) in complex with nirsevimab Fab (gray cartoon) colored by the total effect of mutations at each site on neutralization by nirsevimab Fab. (D) Neutralization curves for nirsevimab IgG or Fab versus pseudovirus expressing the indicated mutants of the subtype A Long strain F. Curves for K68Q and K201T are duplicated from Figure 1. (E) Correlation of mutation effects on…

Figure 4.. Effects of mutations to RSV F on neutralization by clesrovimab IgG and Fab (A) Total decrease in neutralization from all mutations at each site for clesrovimab IgG or Fab as measured by deep mutational scanning. See https://dms-vep.org/RSV_Long_F_DMS/clesrovimab_neutralization.html for interactive plots that show the effects of all mutations. (B) Heatmaps showing effects on neutralization of mutations within the epitope of clesrovimab. Each box represents the effect of a single mutation, with positive values indicating decreased neutralization. Dark gray indicates mutations that are too deleterious for cell entry to measure their effect on neutralization. Light gray shading indicates mutations that were not measured. The wildtype amino acid in the subtype A Long strain used for the deep mutational scanning at each site is indicated with an ‘x’. (C) Pre-fusion RSV F trimer (PDB 6OUS22) in complex with clesrovimab Fab (gray cartoon) colored by the total effect of mutations at each site on neutralization by clesrovimab Fab. (D) Neutralization curves for clesrovimab IgG and Fab of pseudovirus expressing F with point mutations in subtype A (Long) or subtype B (B1) background.…

Figure 5.. Sporadic natural RSV strains have reduced neutralization by nirsevimab or clesrovimab (A) Phylogenetic trees of subtype B RSV F sequences colored by nirsevimab escape scores computed as the summed effects of all mutations as measured by the deep mutational scanning. Strains chosen for validation of nirsevimab neutralization are indicated by boxes and labeled with the Pathoplexus identifier and top resistance mutation. See https://nextstrain.org/rsv/b/F-antibody-escape/6y?c=Nirsevimab-Fab_total_escape for interactive Nextstrain trees that show more sequences and are updated in real time. (B) Tree of subtype A RSV F sequences colored by clesrovimab escape scores. (C) Neutralization curves showing nirsevimab IgG or Fab neutralization of pseudoviruses expressing F from the subtype B strains labeled on the tree. The strains with high escape scores all have reduced neutralization relative to a control strain with a low escape score55. (D) Neutralization curves showing clesrovimab IgG or Fab neutralization of pseudoviruses expressing F from the subtype A strains labeled on the tree. Strains with high escape scores all have reduced neutralization relative to a control strain, except a…

Figure 6.. Effects of mutations to RSV F on neutralization by suptavumab and RSM01 (A) Total decrease in neutralization from all mutations at each site for suptavumab IgG or Fab as measured by deep mutational scanning. See https://dms-vep.org/RSV_Long_F_DMS/suptavumab_neutralization.html for interactive plots that show the effects of all mutations. (B) Heatmaps showing effects on suptavumab neutralization of mutations at top escape sites. Each box represents the effect of a single mutation, with positive values indicating decreased neutralization. Dark gray indicates mutations that are too deleterious for cell entry to measure their effect on neutralization. Light gray shading indicates mutations that were not measured. The wildtype amino acid in the subtype A Long strain used for the deep mutational scanning at each site is indicated with an ‘x’. (C) Pre-fusion RSV F trimer (PDB 5UDC21) colored by the total effect of mutations at each site on neutralization by suptavumab Fab. (D) Phylogenetic trees of subtype B RSV F sequences colored by amino acid identities at sites 172 and 173. (E) Total decrease in neutralization from all mutations at each site for RSM01 IgG or Fab as measured by deep…