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. 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.

Keywords: Genomic Library; Virology.

Graphical abstract

Figure 1

Schematic of Phage-DMS Library Design and Experimental Approach (A) To build a Phage-DMS library, sequences are computationally designed to tile across an entire open reading frame of interest, with the central position varying to contain either the wild-type residue (shown in black) or a mutant residue (shown in red). (B) Sequences are synthesized by releasable DNA microarray and then cloned into a T7 phage display vector. (C and D) (C) The resulting phage display library is incubated with antibody, and (D) phage-antibody complexes are immunoprecipitated with magnetic beads. (E) Sequences from enriched phage are PCR amplified and pooled samples are deeply sequenced. (F) Finally, computational analysis is performed to determine the relative effect of single mutations on the binding of antibody to antigen. Figure made using BioRender.com.

Figure 2

Enrichment and Scaled Differential Selection Results from Phage-DMS for gp41-Specific mAbs with gp41/V3 Libraries (A and B) Line plot showing fold enrichment of wild-type peptides in the background of each HIV Env strain for (A) mAb 240D and (B) mAb F240. (C and D) The color corresponding to each wild-type sequence is shown in the upper right. The x axis shows the amino acid position within HIV Env based on HXB2 reference sequence. (C and D) Heatmap showing the relative effect, as compared with wild-type BG505 Env, of each mutation on the binding to (C) mAb 240D and (D) mAb F240, within a selected region of gp41 shown with HXB2 numbering. Wild-type residues are marked by a black dot. Amino acids are grouped based on their properties as indicated to the left. All data shown are the average of two biological replicates. See Quantification and Statistical Analysis for fold enrichment and scaled differential selection calculations.

Figure 3

Enrichment and Scaled Differential Selection Results from Phage-DMS for V3-Specific mAbs with gp41/V3 Libraries (A and B) Line plot showing fold enrichment of wild-type peptides in the background of each HIV Env strain for (A) mAb 447-52D and (B) mAb 257D. (C and D) The color corresponding to each wild-type sequence is shown in the upper right. The x axis shows the amino acid position within HIV Env based on HXB2 reference sequence. (C and D) Heatmap showing the relative effect, as compared with wild-type BG505 Env, of each mutation on the binding to (C) mAb 447-52D and (D) mAb 257D, within the entire V3 region shown with HXB2 numbering. Wild-type residues are marked by a black dot. Amino acids are grouped based on their properties as indicated to the left. All data shown are the average of two biological replicates. See Quantification and Statistical Analysis for fold enrichment and scaled differential selection calculations.

Figure 4

Enrichment and Scaled Differential Selection Results from Phage-DMS for V3-Specific mAbs with gp120 Libraries (A and B) Line plot showing fold enrichment of wild-type peptides in the background of each HIV Env strain for (A) mAb 447-52D and (B) mAb 257D. (C and D) The color corresponding to each wild-type sequence is shown in the upper right. The x axis shows the amino acid position within HIV Env based on HXB2 reference sequence. (C and D) Heatmap showing the relative effect, as compared with wild-type B41 Env, of each mutation on the binding to (C) mAb 447-52D and (D) mAb 257D, within a selected region of V3 shown with HXB2 numbering. Wild-type residues are marked by a black dot. Amino acids are grouped based on their properties as indicated to the left. All data shown are the average of two biological replicates. See Quantification and Statistical Analysis for fold enrichment and scaled differential selection calculations.

Figure 5

Comparison of Effects of Mutations on Peptide Binding in Competition ELISAs and Phage-DMS (A and B) Bar plot showing the IC50 values of wild-type and mutant peptides in a competition ELISA for (A) gp41-specific mAbs with wells coated with MN gp41 protein or (B) V3-specific mAbs with wells coated with SF162 gp120 protein. Antibodies were pre-incubated with each peptide before addition to the wells. Peptides for which no inhibition of antibody binding could be detected at any concentration tested are indicated with an infinity symbol. Results for three replicate experiments are shown, with the mean ± SEM. Statistical significance was determined by an unpaired t test. (C and D) Correlation between results of IC50 values from competition peptide ELISAs and scaled differential selection values from Phage-DMS for (C) gp41-specific mAbs and (D) V3-specific mAbs. Pearson's correlation shown at the top.