Impact of Respiratory Syncytial Virus season evolution on antibody responses

Abstract

Respiratory Syncytial Virus (RSV) is a leading cause of infant and child mortality, in low-and-middle-income countries. The RSV fusion (F) protein mediates viral entry by undergoing a conformational change from the prefusion (preF) to postfusion (postF) states. Stabilised preF serves as a target for prophylactic or vaccine-elicited neutralising antibodies, and deployment of these RSV preventative interventions may increase selective pressure on the F gene. Immunological surveillance to monitor the functional impact of this diversity on the efficacy of these interventions is crucial. We analysed 569 RSV-A and 370 RSV-B F genomes, identifying 52 antigenic site-specific mutations, 14 of which were frequent across multiple years, increased in frequency in recent years, and/or had predicted antigenic impact. We aimed to develop a F- pseudotyped vesicular stomatitis virus (VSV)-based neutralisation assay to evaluate the impact of these mutations on neutralisation. We produced RSV F-pseudotyped viruses by optimising more than 200 conditions, including multiple cell types and densities, incubation times, F protein C-terminal (CT) modifications, and freeze-thaw parameters. CT modifications increased pseudovirus infection between 9 to 20-fold, with binding of preF and postF mAbs in cell surface expression experiments maintained. However, neutralisation by anti-RSV monoclonal antibodies was not observed likely due to low levels of RSV F protein, mostly in the postF conformation on budded virions. This demonstrates the challenge of establishing an RSV neutralisation assay using pseudotyped VSV particles. Future studies, including a live- virus neutralisation assay using a reverse genetics system and ELISA binding assays, will assess the functional impact of emerging mutations.