Phage-DMS: a comprehensive method for fine mapping of antibody epitopes

Abstract

Understanding the antibody response is critical to developing vaccine and antibody-based therapies and has inspired the recent development of new methods to isolate antibodies. However, methods to define the antibody-antigen interactions that determine specificity or allow escape have not kept pace. We developed Phage-DMS, a method which combines two powerful approachesimmunoprecipitation of phage peptide libraries and deep mutational scanning (DMS) -to enable highthroughput fine mapping of antibody epitopes. As an example, we designed sequences encoding all possible amino acid variants of HIV Envelope to create phage display libraries. Using Phage-DMS, we identified sites of escape predicted using other approaches for four well characterized HIV monoclonal antibodies with known linear epitopes. In some cases, the results of Phage-DMS refined the epitope beyond what was determined in previous studies. This method has the potential to rapidly and comprehensively screen many antibodies in a single experiment to define sites essential for binding to antigen.

High resolution profiling of pathways of escape for SARS-CoV-2 spike-binding antibodies

“…24,820 unique peptides were designed in total; the peptide library included wildtype peptides that could be used to define the antibody epitope and peptides with all possible mutations to determine those within the defined epitope that disrupt or enhance antibody binding. Two biological replicate libraries of these peptide sequences were cloned as we have done previously (Garrett et al, 2020). Deep sequencing of the final duplicate libraries (Library 1 and Library 2) indicated that each contained a high percentage of all unique sequences (96.0% and 95.9%, respectively) ( Figure S1).…”

“…To determine the effect of mutations on antibody binding to S protein epitopes, we compared the relative enrichment of wildtype peptides and mutant peptides within the epitopes defined above. To quantify the effect of each amino acid on binding, we calculated the differential selection of mutant peptides versus wildtype peptides and scaled this value by the strength of binding to the wildtype peptide, as we have previously done for Phage-DMS experiments; this measure is highly correlated with the relative binding of individual mutant peptides by ELISA (Garrett et al, 2020). Plotting the scaled differential selection values for all mutants at each site in a heatmap allows for the visualization of sites where mutations led to a detectable loss of binding.…”

“…After removing duplicate sequences, 24,820 unique sequences were synthesized by Twist Bioscience as an oligonucleotide pool. Two biological duplicate libraries were generated by independently cloning the sequences into a T7 phage vector and then amplifying the phage, as we have done previously (Garrett et al, 2020). Peptides are numbered by the corresponding S protein location of the amino acid in the central position of the peptide.…”

“…Previously we developed a comprehensive method of mapping escape mutations for HIV monoclonal antibodies, referred to as Phage-DMS (Garrett et al, 2020). In Phage-DMS, a library of all possible mutations to a protein is generated in peptide fragments, which are expressed by phage.…”

“…Complexes of antibodies that bind the phage library are immunoprecipitated and sequenced to determine the antibody binding region(s) and the mutations within that epitope region that disrupt binding. Phage-DMS has several advantages: it is high-throughput, allowing comparison amongst a large number of samples in parallel, it can determine pathways of escape for both neutralizing and non-neutralizing antibodies that bind to linear epitopes, and results using this method also correlate well with mutational effects measured in other assays (Garrett et al, 2020). Here, we used Phage-DMS to understand the spectrum of single mutations on the S protein that could reduce antibody binding and thus mediate escape from plasma antibodies found in COVID-19 patients.…”