Clinical Validation of an RSV Neutralization Assay and Testing of Cross-Sectional Sera Pre- and Post- RSV Outbreaks from 2021-2023

Background: Respiratory syncytial virus (RSV) is a leading cause of acute respiratory infections and hospitalization in infants and the elderly. Newly approved vaccines and the prophylactic antibody nirsevimab have heightened interest in RSV immunologic surveillance, necessitating development of high-throughput assays assessing anti-RSV neutralizing activity. Objectives: We validated an RSV focus-reduction neutralization test (RSV FRNT), a high-throughput, automation-ready neutralization assay using RSV strain A2. The assay was then used to investigate the immunity debt hypothesis for resurgent RSV outbreaks in the 2022-2023 season. Study design: We evaluated precision, sensitivity, specificity, linearity, and accuracy of RSV FRNT using reference sera, contrived specimens, and clinical remnant specimens. RSV neutralizing activity of remnant serum specimens, sampled for HSV-1/2 antibody testing during and after the COVID-19 pandemic (February and September 2022 & 2023), was measured and correlated with concurrent trends in RSV prevalence. Results: RSV FRNT was shown to be accurate, generating reference serum neutralizing titers within 2-fold of established assays, with a linear analytical measurement range between 20 to 4,860 ND50 and ND80 units. Neutralizing activity measured with the assay was positively correlated with antibody titer determined via indirect ELISA (ρ = 1.0, p = 0.0014). Among individuals sampled within 3 months of RSV PCR test, RSV positives had a 9.14-fold higher geometric mean neutralizing titer (GMT) relative to RSV PCR negatives (p = 0.09). There was no difference in geometric mean anti-RSV neutralizing titers between sera sampled pre- and post-2023 RSV outbreak (p = 0.93). Conclusions: We validated a high-throughput assay for assessing anti-RSV neutralizing activity and found no significant difference in neutralizing titers between pre- and post-pandemic outbreak specimens.### Competing Interest StatementALG reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen and Hologic and research support from Gilead, outside of the described work.### Funding StatementThis study did not receive any funding### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This study was approved by the University of Washington Medicine Institutional Review Board with consent waiver (STUDY00010205).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors