Fluorescent virus-like particles for investigating humoral responses to SARS- CoV-2 and other respiratory viruses.

SARS-CoV-2 Spike mediates viral entry by shifting between pre- and post-fusion conformations. Pre- and post-fusion Spike is present on the virus, but it remains unclear whether post-fusion Spike is an immune decoy or site of vulnerability. I will screen plasma responses and isolate mAbs against post- fusion Spike. I will examine these mAbs in neutralisation and effector function assays, and investigate the conformational landscape of infected cells with super-resolution microscopy. Waning plasma neutralising antibodies and emerging VOCs increase BTI risk. It is important to generate durable broadly cross-reactive MBC responses, which rapidly reactivate and produce antibodies upon re-exposure. Multiplex staining using molecular barcodes allows in-depth analysis of rare antigen specific B-cells. However, the number of Spike variants studied using this method is often limited. I will incorporate molecular barcodes into fVLPs and integrate them with a custom scRNA-seq platform. This will enable high-throughput analysis of Spike specific MBC breadth across donors with diverse exposure histories, alongside mAb isolation and functional characterisation. The platform will be expanded to investigate additional viral families beyond coronaviruses, forming the basis of a multi-virus deep repertoire analysis approach. Overall, this work will establish fVLPs as a novel approach to investigate humoral responses against viral fusion glycoproteins.