GoKawiilBiosafety 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 infections3,4,6. In the past, escape mutations have been identified from these breakthrough infections or by passaging authentic RSV virus in the presence of antibodies3,4,5,6,14,22,26. 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 nirsevimab13,19, clesrovimab14,40, 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% CO 2 .
Design of deep mutational scanning libraries of RSV F
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