High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike

Abstract

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we established a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrated a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, this method was applied to a region in the S2 domain that includes the first heptad repeat and central helix. Our results revealed that besides K986P and V987P, several mutations simultaneously improved expression and significantly lowered the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

“…We also used the deep mutational scanning system to measure how mutations in spike affect its ability to mediate pseudovirus infection. These measurements complement existing deep mutational scanning datasets on how spike mutations affect the molecular phenotypes of ACE2 affinity (Starr et al, 2022), membrane fusion (Tan et al, 2022), and cell-surface expression (Ouyang et al, 2022;Starr et al, 2022;Tan et al, 2022). None of these experimental measurements fully reflect how mutations affect actual viral fitness, which is an emergent property of many molecular phenotypes (Ballal et al, 2020;Harms and Thornton, 2013).…”

“…There are three points for each mutant, reflecting triplicate measurements. (D) Correlation between enrichment of mutations during actual evolution of human SARS-CoV-2 and functional effects from our lentivirus-based deep mutational scanning or previous RBD expression or ACE2 affinity for yeast-based deep mutational scanning (Starr et al, 2022), and S2 (Tan et al, 2022) or NTD (Ouyang et al, 2022) expression for mammalian display-based deep mutational scanning. Interactive plots that enable mouseovers and show correlations among experiments are at https://dms-vep.github.io/SARS-CoV-2_Omicron_BA.1_spike_DMS_mAbs/natural_enrichment_vs_dms.html…”

“…Our deep mutational scanning measurements of the effects of mutations on spike-mediated infection were reasonably correlated with the enrichment of mutations among actual sequences (Figure 7D), indicating our experiments at least partially reflect the functional selection actually shaping spike evolution. We performed a similar analysis for prior spike deep mutational scanning using yeast display of the RBD (Starr et al, 2022), or mammalian cell display of the NTD (Ouyang et al, 2022) or a region of S2 (Tan et al, 2022) (Figure 7D). Our pseudovirus-based spike deep mutational scanning measurements were more correlated with the enrichment of mutations during actual evolution than any of these prior cell-surface display deep mutational scanning studies, presumably because our experiments mimic the true biological function of spike better than cell-surface display experiments.…”

“…The Illumina barcode sequencing can be found under BioSample accessions SAMN31216920 for Omicron BA.1 and SAMN31230628 for Delta. (Starr et al, 2022), and S2 (Tan et al, 2022) or NTD (Ouyang et al, 2022) expression for mammalian display-based deep mutational scanning. Interactive plots that enable mouseovers and show correlations among experiments are at https://dms-vep.github.io/SARS-CoV-2_Omicron_BA.1_spike_DMS_mAbs/natural_enrichment_vs_dms.html…”

“…However, prior deep mutational scanning of the SARS-CoV-2 spike has been limited to either solely focusing on the receptor-binding domain (RBD) (Cao et al, 2022;Greaney et al, 2021;Starr et al, 2020), other subdomains (Ouyang et al, 2022;Tan et al, 2022) or just a small number of mutations across spike (Javanmardi et al, 2021). Furthermore, all previous spike deep mutational scanning experiments have been based on cell-surface display using either yeast (Starr et al, 2022(Starr et al, , 2020 or mammalian cells (Javanmardi et al, 2021;Ouyang et al, 2022;Tan et al, 2022), and therefore are limited to measuring antibody binding rather than neutralization, despite the fact that neutralization is thought to be a more relevant correlate of protection (Feng et al, 2021;Gilbert et al, 2022).…”