High throughput screening technologies for the SARS-CoV-2 induced syncytium formation

Abstract

Patients of the coronavirus disease 2019 (COVID-19) who acquire signs of severe chronic respiratory diseases frequently experience syncytia, a cell-cell fusion phenomena caused by the Spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Post-mortem patient samples show wide-ranging tissue destruction of lungs along with the formation of massive multinucleated syncytial pneumonocytes. By promoting viral spread, inflammatory response and lymphocyte elimination, syncytia formation is a potential contributing factor to serious pathological consequences. Although it seems technically difficult to screen on a wide scale of Spike protein variants to describe the behavior of cell-cell fusion, a thorough annotation of the ability for Spike protein variants to form syncytia would reveal mutations that could have major pathogenic implications and should be monitored. Here, this thesis work developed a droplet-based microfluidics device and a size-exclusion technique for high-throughput mapping of the Spike variants' capacity to generate syncytium. The syncytium was easily visible under a microscope using the split-GFP complementarity assay, and it can be sorted using fluorescence-assisted cell sorting (FACS). Utilizing these platforms to conduct a thorough mutational analysis of the human cell-bound Spike protein reveals mutable variants at non-receptor binding domain (RBD) regions of the protein, such as the fusion peptide proximity region (FPPR) and furin cleavage site, which augment the Spike protein's capacity to form syncytium. This work verified that the P681H and P681R mutations in the Alpha and Delta SARS-CoV-2 variants are among the mutants that promote the establishment of syncytia. Additionally, the K854H mutant was discovered to confer the Omicron's Spike syncytium-forming potential equivalent to that of the D614G wild-type variety. After calibration, the microfluidics platform provides a high-resolution performance for the study of cell- cell interaction at single-cell level. Conversely, the size-exclusion technique employing a cell strainer offer exceptionally high throughput while compromising a little resolution for the study. These scalable pool-based techniques allow for the quantitative evaluation of cell interaction, which eschew the time-consuming processes involved in creating and analysing genetic mutants one at a time. In the meantime, this thesis work discovered that nanobodies could also mediate the syncytia formation in replacement of the ACE2 receptor, the platform developed was applied to screen a panel of nanobody scaffolds and revealed key residues that contributes to the binding affinity on the nanobody scaffolds. The high-throughput platforms are expected to find extensive utility in the studying cell-cell interactions.