Biosafety and biosecurity

We performed all experiments with pseudotyped lentiviral particles at biosafety level 2 (ref. ⁵⁹). These pseudoviruses do not encode any other viral proteins other than RSV F and therefore can only undergo a single round of cell entry, and so they are not fully replicative infectious agents capable of causing disease. The other viral proteins needed for the formation of pseudotyped lentiviral particles (RSV G, and the lentiviral Gag/Pol, Tat and Rev) were provided during pseudovirus production by transfection of four separate helper plasmids and are not encoded in the viral genome. Therefore, this study did not generate any mutants of fully replicative biological agents capable of causing disease.

Our study quantifies how all single mutations to F affect neutralization by antibodies, including those in clinical use. However, RSV is already evolving under widespread pressure from these antibodies in the human population, with resistant strains regularly identified in breakthrough infections^3,4,6. In the past, escape mutations have been identified from these breakthrough infections or by passaging authentic RSV virus in the presence of antibodies^{3,4,5,6,14,22,26}. Our experiments systematically measure the effects of mutations outside the context of pathogenic virus, and therefore enable informed surveillance of already ongoing evolution on clinical antibodies, as well as informing the design of new antibodies more resilient to viral resistance.

Antibodies

The RSV monoclonal antibodies nirsevimab^13,19, clesrovimab^14,40, palizumab⁴⁷, suptavumab², RSM01 (ref. ¹¹), 1A2 (ref. ⁵²) and 1B6 (ref. ⁵²) were produced by GenScript as human IgG1 kappa isotypes and Fabs. Sequences were obtained from the referenced publications, structures in the Protein Data Bank (PDB) or original patents. Palivizumab originated from patent US6955717B2. See https://github.com/dms-vep/RSV_Long_F_DMS/tree/main/supplemental_data/antibody-sequences for antibody amino acid sequences. The sequence of RSM01 was shared by the Gates Medical Research Institute.

Plasmids and primers

All plasmid and primer sequences used in this study are available via GitHub at https://github.com/dms-vep/RSV_Long_F_DMS/tree/main/supplemental_data. All primers were obtained from Integrated DNA Technologies.

Cell line handling

All cell lines (293T, sourced from the American Type Culture Collection; 293T-TIM1, ref. ³¹ and Takara Lenti-X 293T) were cultured in D10 media (Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U ml⁻¹ penicillin and 100 mg ml⁻¹ streptomycin) and cultured at 37 °C with 5% CO₂.

Design of deep mutational scanning libraries of RSV F

We created pseudovirus libraries containing nearly all possible single amino acid mutations to the ectodomain of the RSV F protein. We used unmutated parental F from the Long strain of RSV, which is a subtype A, laboratory-adapted strain isolated in the 1950s (ref. ³²). We codon optimized this sequence using the GenSmart codon optimization tool offered by GenScript and removed four amino acids from the cytoplasmic tail to increase pseudovirus titres³¹. We used a lentiviral backbone that contains an extended Gag sequence to enhance packaging of lentiviral genomes as previously described in refs. ^60,61. The plasmid map for the lentiviral backbone with codon-optimized RSV F sequence is available from GitHub at https://github.com/dms-vep/RSV_Long_F_DMS/blob/main/supplemental_data/plasmids/4821_v5lp_phru3_forind-extgag_RSV_Long_F_GS4Opt_4aaCTdel.gb.

We aimed to include all single amino acid mutations in the RSV F ectodomain (residues 26–529; 504 × 19 = 9,576 mutations). We also included 30 stop codons located at alternating positions from the start of the ectodomain as a negative control for cell entry measurements. We ordered a site-saturation variant library with these criteria from Twist Biosciences. The final Twist quality control report for the library is available through GitHub at https://github.com/dms-vep/RSV_Long_F_DMS/blob/main/supplemental_data/Final_QC_Report_Q-392996_VariantProportion.csv.

Cloning of deep mutational scanning plasmid libraries of RSV F

The RSV F library was designed to have all mutations to the ectodomain of F. However, 268 mutations were missing from the library produced by Twist. We added the missing mutations and also over-represented mutations in the nirsevimab binding site (amino acid residues 62–69 and 196–212) because we wanted to ensure inclusion and measurement of effects of all possible mutations in the epitope. We aimed to clone a ‘spike-in’ plasmid library that contains these missing mutations and over-representation of mutations in the nirsevimab binding site using a mutagenesis PCR protocol^62,63,64. We designed NNS (where N is any of the four nucleotide bases and S is cytosine or guanine, representing 32 codons that encode all 20 amino acids and a stop codon) primers for the nirsevimab-targeting sites with CodonTilingPrimers (https://github.com/jbloomlab/CodonTilingPrimers) and primers for missing mutations with TargetedTilingPrimers (https://github.com/jbloomlab/TargetedTilingPrimers). Forward and reverse primer pools were created by combining either forward or reverse NNS and targeted mutation primers at an equal molar ratio per codon for a final concentration of 5 µM. Linear template of RSV F was made by digesting the lentiviral backbone with codon-optimized RSV F sequence with NotI-HF and NdeI. PCR mutagenesis was then performed as described previously in ref. ³⁰ with the only difference being that seven PCR cycles were used for the mutagenesis PCR to reduce the resulting number of multi-mutants. After the PCR, the product was digested using DpnI for 20 min at 37 °C to remove any leftover template. The PCR mutagenesis to generate the ‘spike-in’ was performed in duplicate, once for library A and once for library B.

We then barcoded the library pools made by Twist Biosciences independently in two separate reactions to make library A and B, respectively. These two biological replicates were handled separately for all subsequent experimental steps. The barcoding was performed as in ref. ³⁰ using primers containing a random 16 nucleotide sequence downstream of the RSV F stop codon. The only difference from ref. ³⁰ is that only 5 ng of template was used. The two pools of mutagenized RSV F for the spike-in were also barcoded separately. In all there were four separate barcoding PCR reactions. The lentiviral backbone (4016_V5LP_pHrU3_ForInd-Extgag_mcherry)⁶¹ was digested with MluI-HF and XbaI at 37 °C for 45 min then gel purified and purified using AMPure XP beads (Beckman Coulter, A63881). The barcoded libraries were cloned into the lentiviral backbone at a 1:2 insert to vector ratio in a HiFi assembly for 1 h at 50 °C. The HiFi product was purified with AMPure XP beads and eluted in molecular grade water. The purified products were transformed into 10-beta electrocompetent cells (New England Biolabs, C3020K) using a BioRad MicroPulser Electroporator, shocking at 2 kV for 5 ms. Ten reactions of electroporation were performed per barcoded Twist library and two reactions per mutagenized spike-in library. Following the 1 h recovery at 37 °C, transformed cells were spun out of SOC (super optimal broth) and pooled and cultured in 150 ml of Luria-Bertani medium for each Twist library and 100 ml of Luria-Bertani medium for each spike-in library with ampicillin overnight at 37 °C in a shaking incubator. Plasmids were extracted using the QIAGEN HiSpeed Plasmid Maxi Kit (QIAGEN, 12662).

Corresponding replicates of the Twist plasmid libraries and spike-in plasmid libraries were combined at a 1:1.25 Twist to spike-in molar ratio per codon because long-read PacBio sequencing of the plasmid libraries revealed this ratio results in the most even distribution of mutants in the combined libraries.

Production of cell-stored deep mutational scanning libraries

Cells storing the deep mutational scanning libraries as single integrated RSV F containing genomes per cell were produced as described in ref. ³⁰ with a few changes (Extended Data Fig. 1b). VSV-G pseudotyped lentiviruses were produced by transfecting four 10-cm dishes of 293T cells with the lentiviral backbone containing the barcoded RSV F libraries per library (5 μg per dish), lentiviral Gag/Pol, Tat and Rev helper plasmids (1.25 μg per dish, AddGene: HDM-tat1b product ID 204154, pRC-CMV-Rev1b product ID 20413, HDM-Hgpm2 product ID 204152)³⁰ and a VSV-G expression plasmid (1.25 μg per dish, AddGene pMD2.G product ID 12259) using BioT (Bioland Scientific, B01-02).

Whereas previous deep mutational scanning studies have integrated into a specific clone of 293T-rtTA cells that were previously found to yield good pseudovirus titres for other viral entry proteins³⁰, we found that this clone was unable to generate high titre RSV pseudovirus. We tested many clones of 293T-rtTA cells but found that the highest titre RSV pseudovirus produced from singly integrated cells was produced from Takara’s Lenti-X 293T Cell Line (632180). This clonal 293T cell line does not overexpress rtTA. Our lentiviral backbone includes a doxycycline-inducible promoter; in other deep mutational scanning studies that use 293T-rtTA cells and a similar backbone, doxycycline is added to induce expression from the lentiviral backbone³⁰. We found that for production from RSV F cell-stored libraries, pseudovirus titres were sufficiently high when expression was induced only from Tat at transfection. We found no further benefit with transfection of rtTA and addition of doxycycline when producing pseudovirus. Therefore, we used Takara’s Lenti-X 293T Cell Line for the cell-stored library. To make the cell-stored library, the VSV-G pseudotyped viruses were used to infect Takara’s Lenti-X 293T Cell Line at an infection rate of less than 1% so that most transduced cells would receive only a single integrated genome. Transduced cells were selected using puromycin so that the final population of cells contained an integrated genome encoding a single barcoded variant of RSV F. These cells were expanded and frozen with at least 2 × 10⁷ cells per aliquot and stored in liquid nitrogen for later use.

Rescue of F and VSV-G expressing pseudovirus libraries

To rescue F-expressing pseudoviruses from the integrated cells, 100 million cells were plated in 5-layer flasks. The following day, each flask was transfected with 118.75 μg of each helper plasmid (AddGene: HDM-tat1b product ID 204154, pRC-CMV-Rev1b product ID 20413, HDM-Hgpm2 product ID 204152) and 18.75 μg of RSV G expression plasmid (AddGene: HDM_RSV_Long_G_31AACTdel product ID 237350). Xfect transfection reagent (631318) was used according to the manufacturer’s instructions (112.5 μl of Xfect transfection reagent and 7.5 ml of buffer per 5-layer flask). At 48 h after transfection, the supernatant was filtered through a 0.45-μm SFCA Nalgene 500-ml Rapid-Flow filter unit (09-740-44B). Filtered supernatant was then concentrated by ultracentrifugation with a 20% sucrose cushion in Hank’s buffered saline solution (Fisher, 14025092) at 100,000g for 1 h at above 20 °C. The pseudoviruses pelleted to the bottom of the cushion, after disposal of the supernatant, pellets were resuspended in D10. Aliquots of concentrated F-expressing pseudoviruses were flash frozen as previously described in ref. ³¹ and stored at −80 °C for use in downstream selection experiments.

To rescue VSV-G expressing pseudoviruses from integrated cells, 100 million cells were plated in 5-layer flasks. The following day, each flask was transfected with 37.5 μg of each helper (AddGene: HDM-tat1b product ID 204154, pRC-CMV-Rev1b product ID 20413, HDM-Hgpm2 product ID 204152) and VSV-G expression plasmid (AddGene pMD2.G product ID 12259). BioT transfection reagent was used according to the manufacturer’s instructions. At 48 h after transfection, the supernatant was filtered through a 0.45-μm SFCA Nalgene 500-ml Rapid-Flow filter unit and concentrated using Lenti-X concentrator (Takara, 631232) at a 1:3 virus to concentrator ratio, incubating at 4 °C for 3 h and spinning at 1,500g and 4 °C for 45 min. Following centrifugation, supernatant was discarded and viral pellets resuspended in D10. Aliquots of concentrated VSV-G expressing pseudoviruses were frozen at −80 °C for later use.

Long-read PacBio sequencing for variant-barcode linkage

To link the barcode sequences with the mutations found in RSV F we used long-read PacBio sequencing of the lentiviral genomes in pseudoviruses made from the cell-stored libraries. We performed PacBio barcode-mutation linking after generating the singly integrated cell libraries because template switching of the pseudodiploid lentiviral genome during reverse transcription can alter barcode-variant pairings relative to the original plasmid pool. Sequencing the integrated library ensures that barcode-variant linkages reflect those present in the actual integrated provirus. This process has been described in ref. ³⁰. For this library we made a few alterations. A total of 1 × 10⁶ 293T-TIM1 cells were plated in each well of six-well plates coated with poly-l-lysine. The next day, 30 million transducing units (TU) of VSV-G expressing library pseudoviruses were used to infect cells (six wells for each library at 5 million TU per well). At 12 h following infection, the non-integrated reverse-transcribed lentiviral genomes were recovered by miniprepping the 293T-TIM1 cells as described in ref. ³⁰. Amplicons for long-read sequencing of the miniprepped genomes were prepared by following a previously described approach in refs. ^30,64. The PCR reactions for each library were combined and amplicon length was verified by TapeStation before sequencing. Libraries were each sequenced on a single-molecule real-time sequencing cell with a video time length of 30 h on a PacBio Sequel IIe sequencer. To maximize identification of all variants present, each library was sequenced a second time.

We used the dms-vep-pipeline-3 package (https://github.com/dms-vep/dms-vep-pipeline-3), to process sequencing data. To link specific mutations with each barcode, PacBio circular consensus sequences were aligned to the unmutated RSV F reference sequence using the alignparse package⁶⁵. Reads were filtered out if they aligned poorly, had a higher than expected number of mutations in the unmutated regions, did not contain a barcode or were the result of strand exchange. Consensus sequences for each barcode and/or variant sequence were constructed using alignparse, while requiring a minimum of three circular consensus sequence (CCS) reads and a maximum cut off of 0.2 for any minor variants within the consensus. The final barcode and/or variant lookup tables were used as a reference for all downstream analyses of short-read Illumina sequencing of the barcodes only.

The final barcode-variant tables are available from GitHub at https://github.com/dms-vep/RSV_Long_F_DMS/blob/main/results/variants/codon_variants.csv.

For full details on the analysis, see these notebooks for the following:

The final libraries contained 57,888 and 64,078 unique barcoded variants for libraries LibA and LibB, respectively, that covered 99% and 99.4% of all amino acid mutations (Extended Data Fig. 1c). More than half of the variants contain a single amino acid mutation, whereas the others have zero or many mutations (Extended Data Fig. 1d).

Measuring effects of F mutations on cell entry

To measure effects of F mutations on cell entry, we generally followed the approach described in ref. ³⁰ with the following modifications. We infected 293T-TIM1 cells with the pseudovirus libraries showing the RSV F-protein mutants alongside parallel infection of 293T-TIM1 cells with control pseudovirus showing VSV-G to make their cell entry independent of RSV F-protein function (Extended Data Fig. 2a). Next, 2 × 10⁶ 293T-TIM1 cells were plated in each well of six-well plates coated with poly-l-lysine. The next day, we infected cells with roughly 12 × 10⁶ TU total (3 × 10⁶ TU per well) of F pseudovirus library or roughly 3 × 10⁷ TU total (5 × 10⁶ TU per well) of VSV-G pseudovirus library. After addition of pseudovirus, cells infected with the F pseudovirus library were spun at 900g for 3 h at 30 °C. For some selections, 20 μg ml⁻¹ diethylaminoethyl (DEAE)-dextran was added to the media at the time of infection as this increases pseudovirus titres. However, poor cell health was found to contribute to reduced recovery of infecting barcodes, so DEAE was not used for follow-up selections. At 12 h following infection, the non-integrated reverse-transcribed lentiviral genomes were recovered by miniprepping the cells. To prepare the amplicons for Illumina sequencing with dual indexing, PCR was performed as described in ref. ⁶⁴. Samples were pooled in equal DNA amounts and run on a 1% agarose gel. The correct size band was excised, purified with AMPure XP beads, diluted to a concentration of 5 nM and sequenced on an Illumina NextSeq 2000 (with P3 reagent kit) or NovaSeq X Plus system.

We quantified the effects of mutations as the log₂ cell entry of each mutant relative to the unmutated F, using global-epistasis models^33,34 to jointly analyse the single and multi-mutant data. Briefly, Illumina sequencing reads were aligned to the barcode-variant table generated from the PacBio CCS described above. We next compared the frequency of barcodes between the VSV-G and F_mutant selections using the package dms_variants (https://github.com/jbloomlab/dms_variants) as previously described in ref. ³⁰. Cell entry scores for each variant were calculated using log enrichment ratio: log₂ [(n^v_post/n^wt_post)/(n^v_pre/n^wt_pre)], where n^v_post is the count of variant v in the F-pseudotyped infection (post-selection condition), n^v_pre is the count of variant v in the VSV-G-pseudotyped infection (pre-selection condition), and n^wt_post and n^wt_pre are the counts for wild-type variants. Positive cell entry scores indicate that a variant is better at entering the cells compared with the unmutated parental F, and negative scores indicate entry worse than the parental F. As expected, variants with only synonymous mutations had wild-type-like cell entry scores of zero, variants with stop codon mutations had highly negative scores and variants with amino acid mutations had scores ranging from wild-type-like to highly negative (Extended Data Fig. 2b). To calculate the mutation-level cell entry effects, a sigmoid global-epistasis function was fitted to variant entry scores after truncating the values at a lower bound of the median functional score of all variants with stop codons, using the multi-dms software package. For the mutation effects, values of zero mean no effect on cell entry, negative values mean impaired cell entry and positive values mean improved cell entry. To generate the final cell entry effect values for each mutation, we performed a total of three technical replicates for each library, for a total of six functional selections (three each from LibA and LibB). The effects for each mutation were then averaged from the six functional selections. To filter out low quality or noisy data, we required each mutation to occur with two unique barcodes and removed any mutation that had a high standard deviation between replicates. The measured cell entry effects were highly correlated both between replicates and libraries (Extended Data Fig. 2c,d). The final cell entry effect values reported in the figures correspond to the average effect across libraries and replicates.

Measuring effects of F mutations on antibody neutralization

To measure effects of F mutations on antibody neutralization, we used a previously described method from ref. ³⁰ with a few modifications detailed here. A total of 2 × 10⁶ 293T-TIM1 cells were plated in individual wells of poly-l-lysine coated six-well plates. The next day, roughly 1.5–2 × 10⁶ TU of the F pseudovirus library were incubated with D10 media (no-antibody control) or antibody for 1 hour before adding to cells. For monoclonal antibodies, these incubations were performed in a total volume of 2 ml to reduce the possibility of ligand depletion given that these antibodies have very high potency. Antibody concentrations were selected that generally corresponded to a range at which 50% of variants were neutralized, up to 99.5%. During DNA template extraction, we spiked in DNA plasmid containing eight known barcodes that would correspond to roughly 1% of the reads in the no-antibody control. This DNA plasmid spike-in allowed us to estimate the amount of neutralization each antibody condition had relative to the no-antibody control, as previously described in refs. ^30,64 (Extended Data Fig. 4a).

Following Illumina sequencing of the barcodes, the data were analysed as described previously. Briefly, the fraction infectivity retained at each antibody concentration was calculated from the barcode counts of the DNA standard. Then, the polyclonal (https://jbloomlab.github.io/polyclonal/)⁶⁶ software was used to fit neutralization curves and estimates of mutation effects on neutralization. We filtered the effects of mutations on antibody neutralization to retain only mutations with at least two unique barcodes, excluded mutations with very low cell entry scores and excluded mutations that had high standard deviations in their effect on neutralization across replicates. The reported effects of mutations on antibody neutralization are the average across all replicates (always at least two different experimental selections with each of the two independent libraries, LibA and LibB). For example notebooks showing these analyses, see https://dms-vep.org/RSV_Long_F_DMS/notebooks/fit_escape_antibody_escape_RSV-F-LibA-250402-Nir.html for analysis of an individual selection experiment and https://dms-vep.org/RSV_Long_F_DMS/notebooks/avg_escape_antibody_escape_Nirsevimab-IgG.html for average effects across experiments for an antibody.

Validation of cell entry effects using individual pseudoviruses

To validate the effects of mutations on cell entry, we generated a set of plasmids expressing RSV F within a lentiviral backbone that each contained different single F mutations. The parental RSV F amino acid sequence is identical to the unmutated RSV F sequence used in the lentiviral vector described above for deep mutational scanning. We selected mutations spanning a range of entry effects, which included N67I, S215V, S215P, S398L, E87N and D486N. We transfected them into 293T cells, along with helper plasmids (AddGene: HDM-tat1b product ID 204154, pRC-CMV-Rev1b product ID 20413, HDM-Hgpm2 product ID 204152)³⁰ and RSV G in an expression plasmid (AddGene: HDM_RSV_Long_G_31AACTdel Product ID237350). After 48 h, supernatants were filtered through 0.45-μM filters to remove cell debris. The pseudoviruses were titrated on 293T-TIM1 cells using flow cytometry to measure TU per millilitre as previously described in ref. ³¹. Average titres for two replicates of each pseudovirus were then compared to the average unmutated RSV F titre.

Cell entry effects of amino acid differences in natural sequences

An alignment of RSV F-protein sequences for subtype A and B were generated on 27 October 2025 from the RSV Nextstrain workflow⁴⁵. We identified amino acid differences relative to the laboratory-adapted subtype A Long strain used in the deep mutational scanning (for more details see https://github.com/dms-vep/RSV_Long_F_DMS/tree/main/notebooks/sequence_variation). This was used to compare the distribution of cell entry effects for mutations observed in natural sequences to the distribution of cell entry effects for all mutations measured by deep mutational scanning and calculate the percentage of natural sequences with each mutation as shown in Extended Data Fig. 3.

Measuring effects of mutations on antibody neutralization

We chose previously known and newly identified resistance mutations that had a range of effects on antibody neutralization for nirsevimab and clesrovimab. These mutants were chosen to evaluate the impact of mutations in subtype A and B backgrounds on neutralization by antibody IgG and Fab and to validate the effects of mutations measured by deep mutational scanning on antibody neutralization. The single amino acid mutations were made in a subtype A background (Long strain, also used for deep mutational scanning) and a subtype B background (B1 strain) in an expression vector. The plasmids were generated by Twist Biosciences. Pseudoviruses were produced and neutralization was measured as previously described in ref. ³¹. All F constructs included the full cytoplasmic tail and were paired with RSV G in an expression plasmid (AddGene: HDM_RSV_Long_G_31AACTdel Product ID 237350) for these transfections. All plasmid maps can be found through GitHub at https://github.com/dms-vep/RSV_Long_F_DMS/tree/main/supplemental_data/plasmids/. For all plotted neutralization curves, points represent the mean and standard error of at least two replicate measurements.

Mutations affecting nirsevimab neutralization made in subtype A included (N67T, K68N, K68Q, D73N, K201S, K201T, P205S, V207E, K209D, K209Q, Q210T, S211R, S215K) and subtype B (K68N, K68Q, D73N, N201S, N201T, P205S, Q209D, Q210T, S211R, S215K). V207E did not produce usable pseudovirus titres in the B1 background. These are shown in Fig. 3 and Extended Data Fig. 4.

Mutations T67N and Q209K were also made in subtype B, which are mutations to the amino acid residue found in the subtype A Long strain. Data for these mutants along with the corresponding mutations in subtype A (N67T and K209Q) are shown in Extended Data Fig. 4. Subtype A K68Q and K201S and subtype B K68Q and N201S are shown in Figs. 1 and 3 and Extended Data Fig. 4.

The Long and B1 wild-type neutralization curves shown in Fig. 3d,f are representative: all replicates can be found through GitHub at https://github.com/dms-vep/RSV_Long_F_DMS/tree/main/non-pipeline_analyses/validations. The correlation plots in Figs. 3 and 4 show fold-change IC₅₀ from wild type using a matched wild type from the specific experimental date for the mutations and the wild-type point plotted in the correlation plots is a geometric mean IC₅₀ of all wild-type replicates.

Mutations affecting clesrovimab made in subtype A and B included R429M, R429S, S443P and G446D and are shown in Fig. 4 and Extended Data Fig. 6. Pseudoviruses expressing F called ‘A2020’ and ‘B2024’ in Extended Data Fig. 6a are F sequences from natural strains that are broadly representative of recent subtype A and B strains³¹, and have the GenBank accession numbers PP495954.1 and PP660445.1, respectively.

Interactive Nextstrain trees with antibody-escape scores

We integrated computer code into the Nextstrain⁴⁵ RSV view to enable scoring of natural RSV sequences for antibody resistance and visualization of the results on phylogenetic trees. A repository with the computer code implementing this scoring is available from GitHub at http://github.com/nextstrain/rsv. Briefly, the pre-existing Nextstrain view downloaded all RSV sequences from Pathoplexus⁴⁶ (at present, more than 60,000), and built separate subtype A and B phylogenetic trees subsampled to roughly 3,000 sequences designed to be representative across time and countries. We added computer code that assigns each of these sequences an ‘escape score’ calculated from the deep mutational scanning data as either the sum of the effect of all of its constituent mutations or the max effect of any of its mutations on neutralization by the Fab or IgG form of nirsevimab or clesrovimab. The interactive phylogenetic trees can then be coloured by these escape scores using the dropdown ‘Color By’ option on the left toolbar (for example, https://nextstrain.org/rsv/b/F-antibody-escape/6y?c=Nirsevimab-Fab_total_escape); there is also an option to label sequences by their top escape mutation. We integrated this scoring into the Nextstrain RSV builds for the F sequences and the full genome; note that there are also builds that emphasize different timeframes: all-time, the past 6 years and the past 3 years. The builds are updated to include the latest available sequences, so going to views linked above will show the latest sequence data.

We also created new builds called ‘F-antibody-escape’ in the Nextstrain RSV views that shows trees subsampled to include all sequences with high nirsevimab or clesrovimab escape scores. Unlike the ‘F’ builds, these ‘F-antibody-escape’ builds over-represent the frequency of resistant strains. The ‘F-antibody-escape’ trees do not provide an accurate view of the prevalence of resistance mutations; however, when you want to identify all of the top resistant strains (which otherwise could be dropped during subsampling) then these builds should be preferred. Which build is shown can be selected using the ‘change dataset’ option on the left toolbar.

Validation of strains with predicted neutralization resistance

A subset of natural RSV F sequences with predicted resistance to nirsevimab or clesrovimab neutralization identified using the ‘Interactive Nextstrain phylogenetic trees with antibody-escape scores’ were codon optimized and subsequently cloned into an expression vector by Twist Biosciences. F sequences with predicted resistance to nirsevimab Fab or IgG included (PP_002XVQT, PP_002KSRJ, PP_002QMLP, PP_002WHEU, PP_002WWH8, PP_002SUFP, PP_001QYN9, PP_001Y2UB, PP_003W55P). Sequences with predicted resistance to clesrovimab Fab or IgG included (PP_002W1BG, PP_001WGC0, PP_001Y62S, PP_001ZQ7W). PP_001W26S and PP_002UDSB correspond to subtype A and B controls also referred to in our previous study as A2020 and B2024, respectively³¹. A complete list of these key sequences are available in Pathoplexus under SeqSet PP_SS_628.1 (ref. ⁶⁷) and all sequences shown in the trees in Fig. 5 and Extended Data Fig. 8 are available in Pathoplexus under SeqSet PP_SS_661.1 (ref. ⁶⁸) for subtype A and SeqSet PP_SS_662.1 (ref. ⁶⁹) for subtype B. All plasmid maps can be found through GitHub (https://github.com/dms-vep/RSV_Long_F_DMS/tree/main/supplemental_data/plasmids/strain%20validations). These were used to generate RSV pseudoviruses expressing the F from the natural sequences paired with RSV G expression plasmid (AddGene: HDM_RSV_Long_G_31AACTdel Product ID237350) as previously described in ref. ³¹. Producing RSV pseudoviruses with F from natural sequences paired with all the same G ensures we do not introduce any variability from G. These pseudoviruses were then used in neutralization assays with nirsevimab or clesrovimab IgG and Fab³¹. To ensure reliable neutralization curves, we established a cut off of 400,000 relative light units per well for the no-antibody, virus-only control. For strains with titres near the cut off, extra controls were included to ensure escape was specific to the monoclonal antibody and not attributable to experimental artefacts.

Structural analysis

UCSF ChimaX⁷⁰ was used for structural visualizations. All PDB accession IDs used are included in figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.