Genetic variability of respiratory syncytial virus and its impact on monoclonal antibody binding sites: a national cross-sectional study during the 2023-2024 season

Abstract

Background: Respiratory syncytial virus is a primary cause of acute lower respiratory tract infections globally. As preventive tools such as vaccines and monoclonal antibodies begin to enter clinical use, baseline genomic data are critical to evaluate their future impact and detect potential resistance-related mutations. The working group on respiratory viral infections (GLIViRe) conducted this multicenter study to characterize the genetic profile of RSV circulating in Italy during the 2023-2024 season, immediately prior to the introduction of immunoprophylactic interventions. The study focused on identifying mutations in the F protein at mAb binding sites for palivizumab, nirsevimab, RSM01, TNM-001, and clesrovimab.

Methods: A total of 350 respiratory samples positive for RSV collected from patients with influenza-like illness (ILI) or acute respiratory infection (ARI), during the 2023-2024 season from 15 Italian laboratories were selected for sequence analysis. The F gene sequencing was performed on 287 RSV-A and 63 RSV-B samples using Sanger or next-generation sequencing. Phylogenetic analysis was conducted using IQ-TREE, with the integration of global data via NextStrain. Key mutations were mapped onto the F protein structure using ChimeraX and Protein Data Bank models. Shannon entropy was used to assess amino acid variability.

Results: RSV-A samples predominantly belonged to the emerging A.D, A.D.1, and A.D.3 clades, while RSV-B samples mainly clustered in the B·D lineage. Key substitutions were detected at antigenic site ∅, particularly at the nirsevimab and RSM01 interfaces. No changes occurred at the palivizumab/TNM-001 site II. All mutations of interest were exposed to the F protein surface.

Conclusions: This study provides a critical genomic snapshot of RSV in Italy prior to the introduction of vaccines and mAbs. Continuous surveillance is essential for monitoring viral evolution and supporting the long-term effectiveness of future immunization strategies.

Keywords: Molecular evolution, monoclonal antibodies; Nirsevimab; Palivizumab.; Prophylaxis; Respiratory syncytial virus.