Abstract Background Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection (LRTI) and hospitalization in infants. In two global pivotal placebo-controlled studies, nirsevimab, a monoclonal antibody to the RSV prefusion (F) protein with extended half-life, reduced medically attended (MA) RSV LRTI versus placebo throughout the RSV season (MELODY (Primary Cohort)/Study 3 (Proposed Dose) Pool, 79.5% efficacy). Here we summarize resistance analyses of all RT-PCR-confirmed RSV isolates from healthy term and preterm infants through 150 days post dose. Methods Infants were randomized 2:1 to receive one intramuscular injection of nirsevimab or placebo, prior to their first RSV season. RT-PCR-confirmed RSV isolates were reflexed for genotypic analyses of RSV F and phenotypic analyses of identified substitutions in a recombinant RSV neutralization susceptibility assay. Results In the pooled proposed dose analysis of Study 3 (50 mg nirsevimab if < 5 kg at dosing) and MELODY (50 or 100 mg nirsevimab if < 5 kg or ≥5 kg at dosing, respectively), no subject with MA RSV LRTI had an RSV isolate containing nirsevimab resistance-associated substitutions in either treatment group (nirsevimab, RSV A: 0/14 and RSV B: 0/5; placebo, RSV A: 0/35 and RSV B: 0/16). In Study 3 (50 mg nirsevimab if ≥5 kg at dosing), 2/18 subjects in the nirsevimab group and 0/20 subjects in the placebo group with MA RSV LRTI had an RSV isolate harbouring nirsevimab binding site substitutions I64T+K68E+I206M+Q209R (>447-fold) or N208S (>387-fold) that conferred reduced susceptibility to nirsevimab neutralization (nirsevimab, RSV A: 0/9 and RSV B: 2/9; placebo, RSV A: 0/10 and RSV B: 0/10). Subjects with RSV isolates harboring F protein sequence variations that maintained susceptibility to nirsevimab neutralization were balanced between treatment groups with no association with RSV disease severity. No subjects with non-protocol defined MA RSV LRTI cases or hospitalization due to any RSV respiratory illness had an RSV isolate conferring nirsevimab resistance. Conclusion Lack of nirsevimab resistance following immunization at the proposed dose supports efficacy and neutralization activity of nirsevimab against both RSV A and B strains throughout the RSV season. Disclosures Michael E. Abram, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Bahar Ahani, BSC, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds David E. Tabor, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Fiona Fernandes, PhD, AstraZeneca: Stocks/Bonds Deidre Wilkins, BSC, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Anastasia A. Aksyuk, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Kevin M. Tuffy, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Hong Ji, BSc, AstraZeneca: Stocks/Bonds Christine Blaze, BSc, AstraZeneca: Stocks/Bonds Tyler Brady, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Pamela Griffin, MD, AstraZeneca: Stocks/Bonds Amanda Leach, MD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Tonya L. Villafana, PhD, MPH, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Stocks/Bonds.