GSE159372
An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants
The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry.  Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape.  Here, we directly test this hypothesis.  Using deep mutagenesis, we find that the ACE2-binding surface of the SARS-CoV-2 spike tolerates high mutational diversity, which may act as a source for resistance to therapeutics.  However, saturation mutagenesis of the receptor-binding domain (RBD) followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor.  We conclude that resistance to engineered decoys will be rare. Overall design: A human codon optimized gene for full-length SARS-CoV-2 S (GenBank YP_009724390.1) with an N-terminal c-myc tag was diversified by site-saturation mutagenesis across the RBD (a.a. C336-L517).  Degenerate NNK codons were introduced at each residue position to provide a library that scans the RBD sequence of S. The library was expressed in Expi293F cells that were then sorted by FACS in two different ways.  First, the library was incubated with wild type sACE2, and cells were collected that both expressed myc-tagged S and either bound sACE2 at high or low levels, referred to as the ACE2-High and ACE2-Low sorts, respectively.  Second, the library was incubated with competing wild type sACE2 and engineered sACE2.v2.4, which binds the SARS-CoV-2 spike with high affinity.  Now, cells were collected where binding signals were shifted to favor the binding of wild type or engineered receptors, referred to as the WT-specific and v2.4-specific sorts.  The specificity selections were replicated in an independent experiment.  RNA was extracted from the sorted cell populations and Illumina sequenced. Transcript frequencies for all substitutions within the RBD were compared to their respective frequencies in the naive plasmid library to calculate an enrichment ratio.