SARS-CoV-2 Spike Protein Engineering

The Δ19 spike clone with the D614G mutation, as previously described [10 (link)], was used as a starting template to generate the 22-10-04 spike and was digested using the restriction enzymes SacII and XhoI (New England Biolabs). The 12 missense mutations found in 22-10-04 were designed using A Plasmid Editor (ApE [11 ]), ordered as a gBlock (Integrated DNA Technologies), and inserted into the D614G Δ19 spike vector at the SacII and XhoI sites using the 5x In-Fusion Snap Assembly Master Mix (Takara Bio). The 22-10-04 clone was confirmed via Sanger Sequencing (Genomics Technology Core - University of Missouri). The vector for HIV-1-Gluc particles was previously described [12 (link)]. The GFP-N1 plasmid was originally provided by Clontech. All mAb plasmids were initially obtained as 4 μg maxipreps from Genscript, transformed into DH5α cells, and then isolated using the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen). The plasmids for bamlanivimab included LY_CoV555_HC_pcDNA3.4 and LY_CoV555_LC_pcDNA3.4. The plasmids for etesevimab included CB6_HC_pcDNA3.4 and CB6_LC_pcDNA3.4. The plasmids for casirivimab included REGN10933_HC_pcDNA3.4 and REGN10933_LC_pcDNA3.4. The plasmids for imdevimab included REGN10987_HC_pcDNA3.4 and REGN10987_LC_pcDNA3.4.