A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

Abstract

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~'10 to the 5' combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection

“…To elucidate the epitope of C68.59, which does not bind RBD and thus is not a candidate for the yeast RBD display system, we used a pseudotyped lentivirus DMS system with a library covering functionally tolerated mutations across the entire spike glycoprotein (66). In this assay, escape is measured by comparing the mutant pseudotyped viruses that infect in the presence versus absence of the antibody.…”

“…The escape conferred by each mutation was determined relative to a VSV-G pseudotyped neutralization standard as described in (66). This analysis used the biophysical model implemented in polyclonal package (https://jbloomlab.github.io/polyclonal/) (66). The computational pipeline used to implement this analysis is at…”

“…Full BA.1 spike lentivirus-based deep mutational scanning libraries were made as described previously (66). In brief, the BA.1 spike deep mutational scanning libraries are designed to contain all functionally tolerated mutations at every position in spike.…”

A biophysical model of viral escape from polyclonal antibodies

“…2021c ). While previous studies have been restricted to variants that predominantly contain single amino acid mutations, recent advances enable combinations of mutations to be assayed as well ( Dadonaite et al 2022 ). Although only an infinitesimal fraction of all combinations of mutations can be assayed, this approach does measure \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$p\left( {v,c} \right)$\end{document} for each mutation in many different backgrounds, which should be sufficient for revealing the epitopes targeted by polyclonal antibody mixtures and their associated escape mutations.…”

“…Despite these limitations, our model not only predicts the escape potential of viral variants with arbitrary combinations of mutations that are observed in a simulated deep mutational scanning library—it also clarifies how escape mutations combine to determine the magnitude of viral escape. While this manuscript was under review, our model was successfully applied to real deep mutational scanning data measuring the escape of SARS-CoV-2 spike variants from monoclonal antibodies, yielding predictions that strongly correlated with neutralization assays ( Dadonaite et al 2022 ). While this paper only considers simulated data, we envision that our model can be further applied to appropriately designed deep mutational scanning experiments to address two main questions: (1) delineating the epitopes targeted by polyclonal serum and (2) predicting the antigenic properties of new variants with arbitrary combinations of mutations.…”