Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes
clinical diagnostics molecular biology structural biology
Authors:Zhou et al.
Link to paper:https://doi.org/10.1101/2020.06.22.166033
Tags: Clinical/ Diagnostics, Molecular Biology, Structural Biology
Molecular probes mimicking SARS-CoV-2 Spike protein have been produced and characterised for antigenicity, ACE recognition, antibody-binding specificities and cell-sorting capabilities.
The structure of the spike ectodomain probe was determined by cryo-electron microscopy and was found to be very similar to the spike ectodomain of the SARS-COV-2 virus.
Produced three RBD probes with 2 or 3 mutations which completely ablated ACE2 binding in order to distinguish between antibodies which target the ACE2 binding site and other epitopes on the RBD.
Molecular probes mimicking full-length SARS-CoV-2 spike protein ectodomain as well as various sub-regions were produced by tag-based purification with biotinylation and HRV3C protease cleavage, and characterised for antigenicity and ACE2 recognition. They also developed mutant RBD probes which distinguish antibodies binding at the ACE2 interacting site and other epitopes on the RBD. The probes had good antibody-binding specificities and were shown to be able to be used on SPR, and to sort both yeast cells expressing SARS-CoV-2 spike-binding antibodies and B cells from a COVID-19 convalescent donor. The structure of the spike ectodomain probe was determined by cryo-electron microscopy and showed good similarity with the reported protein structure. These probes show potential for vaccine development and developing antibody therapeutics.
Impact for SARS-CoV2/COVID19 research efforts
Others: Developed a tool which could assist in monitoring antibody responses to vaccination as well as characterising antibodies
In vitro study
Strengths and limitations of the paper
Novelty: Molecular SARS-CoV-2 antigen probes produced can be used both on SPR and in sorting Yeast and B cells. The structures of the probes were solved by cryo-EM. The RBD mutant probes are able to distinguish between antibodies which directly target the ACE2 binding site and those which bind to other epitopes.
Standing in the field:The structures of the probes are mostly consistent with the reported S trimer and subdomain structures.
Appropriate statistics:Statistics are accurate
Viral model used:SARS-CoV-2 antigens but with stabilising mutations
Translatability:These probes show high potential for vaccine development and antibody therapeutic development.
S probe designs reported here are similar to other published soluble S trimers and their subdomains, and the structural analysis is broadly confirmatory of other studies.
The yield of the probes mimicking the full-length SARS-CoV-2 spike protein ectodomain is much lower than the ones mimicking the subdomains. Is this yield good enough to be streamlined?