The true variety of replicates where escape variants were selected are indicated, color coded according to whether escape was selected frequently (red) or rarely (white). of antibody assessment and therapeutics from the antigenic consequences of viral evolution. == Launch == The COVID-19 pandemic provides generated urgent curiosity about antibody therapeutics and vaccines that creates antibodies to SARS-CoV-2. Some of the Hesperidin most potently neutralizing anti-SARS-CoV-2 antibodies focus on the receptor-binding area (RBD) from the viral spike proteins, often competing using its binding towards the ACE2 receptor (Brouwer et al., 2020;Cao et al., 2020;Ju et al., 2020;Liu et al., 2020;Rogers et al., 2020;Seydoux et al., 2020;Wec et al., 2020;Wu et al., 2020;Zost et al., 2020a,2020b). Furthermore, anti-RBD antibodies frequently dominate the neutralizing activity of the polyclonal antibody response elicited by organic infections (Barnes et al., 2020a;Steffen et al., 2020;Weisblum et al., 2020). Both passively-administered and vaccine-induced anti-RBD neutralizing antibodies drive back SARS-CoV-2 in pets (Alsoussi et al., 2020;Cao et al., 2020;Hassan et al., 2020;Rogers et al., 2020;Wall space et al., 2020a;Wu et al., 2020;Zost et al., 2020a), and primary evidence suggests the current presence of neutralizing antibodies also correlates with Hesperidin security in human beings (Addetia et al., 2020). Identifying which viral mutations get away from antibodies is essential for creating therapeutics and vaccines and evaluating the antigenic implications of viral progression. Escape mutants could be chosen KLF5 by passaging pathogen expressing the SARS-CoV-2 spike proteins in the current presence of anti-RBD antibodies in the lab (Baum et al., 2020a;Weisblum et al., 2020), plus some RBD mutations that alter antibody binding Hesperidin already are present at suprisingly low amounts in SARS-CoV-2 circulating in the population (Li et al., 2020). It appears plausible that such mutations could become widespread over evolutionary period much longer, considering that the seasonal coronavirus 229E provides accumulated genetic deviation in its RBD within the last few years that’s enough to ablate antibody binding (Wong et al., 2017). Nevertheless, current solutions to recognize SARS-CoV-2 get away mutations by passaging pathogen in the current presence of antibodies are imperfect in the sense that they only find one or a few of the possible escape mutations. Structural biology can more comprehensively define how an antibody physically contacts Hesperidin the virus, but structures are time consuming to determine and still do not directly report which viral mutations escape from antibody binding (DallAcqua et al., 1998;Dingens et al., 2019;Jin et al., 1992). Here we overcome these limitations by developing a high-throughput approach to completely map mutations in the SARS-CoV-2 RBD that escape antibody binding, and apply this approach to 10 human antibodies. The resulting escape maps reveal the extent to which different antibodies are escaped by mutations at overlapping or orthogonal sites, and show that antibodies targeting structurally similar regions sometimes have escape mutations at entirely distinct residues. Furthermore, we show that the escape maps predict which mutations are selected when spike-expressing virus is passaged in the presence of neutralizing antibodies, and can inform the design of antibody cocktails that resist escape. Therefore, complete escape-mutation maps can be used to assess the antigenic consequences of viral genetic variation and the potential for viral escape from specific antibodies or antibody cocktails. == Results == == A yeast-display system to completely map SARS-CoV-2 RBD antibody-escape mutations == To map antibody-escape mutations in a high-throughput manner, we leveraged a system for expressing conformationally-intact RBD on the surface of yeast cells (Figure 1A). As described previously (Starr et al., 2020), we created duplicate mutant libraries of the RBD from the Wuhan-Hu-1 strain of SARS-CoV-2 that together contained nearly all possible amino-acid mutations in the 201-residue RBD (they contain 3,804 of the 3,819 possible mutations, with >95% present as single mutants). Each yeast cell carries a short 16-nucleotide barcode that identifies the RBD mutant it expresses, enabling us to rapidly characterize the composition of the RBD mutant libraries via deep sequencing of the DNA barcodes. == Figure 1. A yeast-display system to completely map SARS-CoV-2 RBD antibody escape mutations. == (A) Yeast display RBD on their surface. The RBD contains a c-myc tag, enabling dual-fluorescent labeling to quantify both RBD expression and antibody binding of RBD by flow cytometry. (B) Per-cell RBD expression and antibody binding as measured by flow cytometry for yeast expressing unmutated RBD and one of the RBD mutant libraries. (C) Experimental workflow. Yeast expressing RBD mutant libraries.