A key difference is that for the previously published Wuhan-Hu-1 experiments, dimeric rather than monomeric ACE2 was used [25]. (EPS) Click here for additional data file.(6.8M, eps) S3 FigDeep mutational scanning approach to map mutations that reduce binding of B.1.351 infection-elicited polyclonal plasma antibodies to the B.1.351 RBD.(A) Schematic of the approach. did not abrogate binding to RBD. There were not substantial reductions in OD450 after the third round of depletions, so we reasoned that this samples were maximally depleted, and no further rounds of depletions CID16020046 were performed. Removal of RBD-binding antibodies only modestly reduces spike binding, consistent with prior findings that the majority of anti-spike antibodies do not bind the RBD [28,77C80]. (C) Neutralization curves for plasma mock depletion (gray circles) and depletion of RBD-binding antibodies (orange triangles). Each assay was performed in technical duplicate, and points show the mean and standard error of the replicates. Pre-pandemic pooled serum was included in (B) and (C) as a negative control for binding and neutralization. RBD-binding antibodies were removed from the plasma using streptavidin magnetic beads conjugated to biotinylated B.1.351 RBD. All binding assays were performed with B.1.351 RBD CID16020046 and spike, and all neutralization assays were performed with B.1.351 spike-pseudotyped lentiviral particles. This figure shows the underlying measurements for all of the B.1.351 plasmas in Fig 2; the underlying measurements for the early 2020 plasmas in Fig 2 are shown in [28].(EPS) ppat.1010248.s001.eps (2.9M) GUID:?80E5CE3A-EB04-4807-B773-2535D82F0139 S2 Fig: Generation of the B.1.351 RBD CID16020046 mutant libraries and measurements of effects of mutations Rabbit Polyclonal to GRB2 on ACE2 binding and RBD expression. (A) Schematic showing the B.1.351 RBD mutant library design. A site-saturation variant library was generated in the B.1.351 RBD background, targeting one amino-acid mutation per variant. Nx16 unique DNA barcodes were added to the variant gene fragments. The Nx16 barcodes were linked to their associated RBD mutations by PacBio circular consensus sequencing (CCS). The plasmid library DNA was transformed into yeast cells. In downstream experiments, the Nx16 barcodes are sequenced by short-read Illumina sequencing. The tables at right indicate key library statistics. (B) Correlations CID16020046 between biological independent replicate library measurements of the effects of single mutations on ACE2 binding and RBD expression, measured as described in [25]. See Methods for experimental details. (C) Thresholds around the ACE2 binding and RBD expression scores (dashed orange lines) for the B.1.351 mutant library to computationally filter highly deleterious variants that may represent spurious antibody-escape mutations. Importantly, we aimed to retain most mutations that have been observed > = 50 occasions in sequenced SARS-CoV-2 isolates. The x-axis categorizes mutations by their number of observations in GISAID [81] as of Aug. 1, 2021. An ACE2 binding score threshold of > = C3.0 (1,000-fold loss in binding affinity) and an RBD expression score of > = C1.0 (10-fold loss in RBD expression) were chosen, which filter comparable numbers of mutations as in prior Wuhan-Hu-1 experiments [28,73]. These filters retain 99.4 and 93.8% of mutations, respectively, that have been observed > = 50 times in sequenced SARS-CoV-2 isolates. (D) Relationship between the ACE2 binding and RBD expression scores for the B.1.351 RBD library compared to those CID16020046 previously published for the Wuhan-Hu-1 library [25]. The computational filters used for antibody-escape experiments for the Wuhan-Hu-1 [28] and B.1.351 libraries are dashed orange lines. Each dot is usually one mutation, and mutations to disulfide bonds are shown in red. A key difference is usually that for the previously published Wuhan-Hu-1 experiments, dimeric rather than monomeric ACE2 was used [25].(EPS) ppat.1010248.s002.eps (6.8M) GUID:?3F623BAC-11BB-45DC-A67F-A2714B836A57 S3 Fig: Deep mutational scanning approach to map mutations that reduce binding of B.1.351 infection-elicited polyclonal plasma antibodies to the B.1.351 RBD. (A) Schematic of the approach. The RBD is usually expressed on the surface of yeast (top left). Flow cytometry is used to quantify both RBD expression (via a C-terminal MYC tag, green star) and antibody binding to the RBD protein expressed on the surface of each yeast cell (bottom left). A library of yeast expressing B.1.351 RBD mutants was incubated with convalescent plasmas and fluorescence-activated cell sorting (FACS) was used to enrich for cells expressing RBD that bound reduced amounts of plasma antibodies, as detected using an IgA+IgG+IgM secondary antibody. Deep sequencing was used to quantify the frequency of each mutation in the initial and antibody-escape cell populations. We quantified the effect of each mutation as the escape fraction, which represents the fraction of cells expressing RBD with that mutation that fell in the antibody escape FACS bin. Escape fractions are represented in logo plots, with the height.