Phage-DMS: A Comprehensive Method for Fine Mapping of Antibody Epitopes

Abstract

Summary 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. Methods to define the antibody-antigen interactions that determine specificity or allow escape have not kept pace. We developed Phage-DMS, a method that combines two powerful approaches—immunoprecipitation of phage peptide libraries and deep mutational scanning (DMS)—to enable high-throughput fine mapping of antibody epitopes. As an example, we designed sequences encoding all possible amino acid variants of HIV Envelope to create phage 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 interactions.

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies

“…Lineage 105 targets the V3 loop and there were changes in this region among the QA255 Env sequences, although it was unknown if the sequence changes would impact binding of 105 to its epitope. To address this, we finely mapped the epitope and sites of escape for both the mature and naive mAbs using a phage-display deep mutational scanning (Phage-DMS) approach ( Garrett et al, 2020 ). The mAbs were screened using a library displaying gp41 and V3 peptides from HIV strains BG505.W6M.C2 (clade A), BF520.W14M.C2 (clade A), and C.ZA.1197MB (clade C).…”

“…Profiling of escape mutations was done as previously described ( Garrett et al, 2020 ). We utilized a deep mutational scanning (DMS) phage display library containing wildtype and mutant peptides that tile across the gp41 and V3 portions of three Envelope strains (BG505.W6M.C2, BF520.W14M.C2, and C.ZA.1197MB).…”

“…RFADCC experimental exclusion criteria are detailed within the RFADCC method section above. For Phage-DMS, calculations were performed as previously described ( Garrett et al, 2020 ), using RStudio; Phage-DMS analysis source code is available in Figure 9—source code 1 .…”

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).…”

“…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.…”