High-resolution epitope mapping by AllerScan reveals relationships between IgE and IgG repertoires during peanut oral immunotherapy

Abstract

Peanut allergy can result in life-threatening reactions and is a major public health concern. Oral immunotherapy (OIT) induces desensitization to food allergens through administration of increasing amounts of allergen. To dissect peanut-specific immunoglobulin E (IgE) and IgG responses in subjects undergoing OIT, we have developed AllerScan, a method that leverages phage-display and next-generation sequencing to identify the epitope targets of peanut-specific antibodies. We observe a striking diversification and boosting of the peanut-specific IgG repertoire after OIT and a reduction in pre-existing IgE levels against individual epitopes. High-resolution epitope mapping reveals shared recognition of public epitopes in Ara h 1, 2, 3, and 7. In individual subjects, OIT-induced IgG specificities overlap extensively with IgE and exhibit strikingly similar antibody footprints, suggesting related clonal lineages or convergent evolution of peanut-specific IgE and IgG B cells. Individual differences in epitope recognition identified via AllerScan could inform safer and more effective personalized immunotherapy.

Trial registration: ClinicalTrials.gov NCT01781637.

Keywords: IgE; allergen immunotherapy; antibody; antibody repertoire; epitope mapping; food allergy; high-throughput sequencing; oral immunotherapy; peanut allergy; phage display.

Conflict of interest statement

S.J.E. is a co-founder of TSCAN Therapeutics, MAZE Therapeutics, ImmuneID, and Mirimus. S.J.E. serves on the Scientific Advisory Board (SAB) of CRISPR Therapeutics; Homology Medicines, Inc.; TSCAN Therapeutics; and X-Chem and is an advisor for MPM. S.J.E. and G.C. have applied for a patent on these findings. J.M.S. has received research funding from Aimmune Therapeutics, Regeneron, Sanofi, Novartis, and FARE. K.C.N. is co-founder of IgGenix, Latitude, Alladapt, and BeforeBrands. She also serves on the SAB or Data and Safety Monitoring Boards (DSMB) of Novartis, Regeneron, ClostraBio, Cour Pharmaceuticals, Aravax, and NHLBI. D.T.U. and H.C.O. serve on the SAB of IgGenix. J.A.P. is on the Independent Data Monitoring Committee of Regeneron and Clinical Advisory Board of FARE and has served as an investigator for Aimmune Therapeutics and FARE. She has received research funding from DBV, FARE, The Sunshine Charitable Foundation, and Peak6 Investments LLC. A.J.M. is an advisor to DBV Technologies. R.R. has received research funding from Aimmune Therapeutics. W.P. is a consultant for Genentech, Novartis, Regeneron, and Sanofi and received research support from Thermo Fisher Scientific. L.S. serves on the Medical Advisory Board for Food Allergy Research and Education and the DSMB for Alladapt and is an investigator for DBV Technologies and Regeneron. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript.

© 2021 The Author(s).

Figures

Graphical abstract

Figure 1

AllerScan reveals epitope targets of anti-peanut IgE and IgG antibodies in allergic patients and controls (A) Construction of peanut AllerScan library and overview of the AllerScan workflow. The peanut AllerScan library consists of 20-mer peptides tiling across the sequences of all peanut allergen proteins as well as saturating mutant versions of the peptides. DNA oligonucleotides encoding these peptides were synthesized and cloned into a phage-display library. To perform an AllerScan reaction, serum is mixed with the AllerScan phage library, IgE or IgG antibodies are immunoprecipitated, and bound phage is sequenced to identify the displayed peptides. (B) Distribution of the peanut AllerScan library. At approximately 60-fold sequencing coverage, 99.34% of library members were detected and 74.8% were within one-log abundance. (C) Heatmaps depict the IgE (blue) and IgG (red) antibody response to peanut peptides in allergic patients (n = 15) and healthy controls (n = 30 for IgE; n = 54 for IgG). Each row represents a sample from a unique individual. Each column represents a wild-type peptide from the peanut allergen protein labeled at top. The color intensity indicates the Z score representing the level of enrichment of the peptide. Z scores are averages of 2 replicates. (D) Seroprevalence of antibodies to peanut epitopes among allergic individuals. y axis shows percentage of allergic patients exhibiting IgE (blue) and IgG (red) reactivity to peptides in the peanut AllerScan library. Baseline allergic sera were collected at week 0 of the PRROTECT study.

Figure 2

Mapping of IgE and IgG epitopes in major peanut allergens using saturation mutants Each column of the heatmap corresponds to an amino acid position; each row represents an allergic patient. The color intensity indicates the substitution effect at each amino acid position within the peanut allergens, blue representing IgE epitopes and red representing IgG epitopes (details in STAR Methods). Deeper shades represent greater disruption of antibody binding. For simplicity of presentation, results of only one of the technical replicates are shown.

Figure 3

High-resolution profiling of antibody footprints reveals conserved public peanut epitopes (A–C) Representative examples of high-resolution antibody footprints from allergic patients (week 0 samples) for public peanut epitopes Ara h 2.02 aa21–40 (A), Ara h 3.01 aa301–320 (B), and Ara h 7.01 aa71–90 (C). Heatmaps plot the −log10 transformed relative enrichment compared to the adjusted wild-type value, which represents the substitution effects on antibody binding. x axis, amino acid sequence of the wild-type peanut epitope; y axis, amino acid substitutions. Critical residues for antibody binding are indicated at the top of each heatmap in blue; non-critical residues are indicated in red. (D–F) Pairwise Pearson correlations between high-resolution footprints of all allergic patients with antibody responses to the three peptides from (A)–(C), respectively. (G) Number of patients who share the dominant IgE antibody footprint for the public epitopes indicated by the x axis. Blue, patients who share the dominant footprint; white, patients with non-dominant footprints.

Figure 4

Peanut oral immunotherapy diversifies peanut-specific IgG repertoire (A) Heatmaps depict IgE (blue) or IgG (red) binding signal to individual wild-type peptides before (week 0, upper) and after (week 52, lower) OIT. Patients were stratified into 3 categories, depending on the type of IgE and IgG binding change. Top: patients exhibiting overall IgE binding decrease and IgG increase are shown; middle: patients with unchanged IgE but increased IgG are shown; bottom: patients with mixed changes in IgE and IgG binding are shown. (B) Boxplots depict the number of IgE (blue) and IgG (red) peptides recognized by each allergic patient before and after OIT (n = 15 biological replicates). Only peptides from 1 variant of each Ara h protein were included in the calculation. Wilcoxon signed rank test was used to determine statistical significance. (C) Jaccard index representing IgE and IgG repertoire overlap at week 0 and week 52 of OIT. p value indicated on top was calculated using Wilcoxon matched-pair signed rank test. (D) Overlap between IgE and IgG epitopes at week 52 of OIT. y axis shows each of the following categories in fraction of total epitope: blue, epitopes exclusively recognized by the IgE; red, epitopes exclusively recognized by IgG; brown, epitopes recognized by both the IgE and IgG.

Figure 5

Peanut oral immunotherapy increases peptide-specific IgG levels while reducing abundance of pre-existing IgE specificities (A) Change in seroprevalence of IgE (left) and IgG (right) against individual epitopes after OIT. Ara h 7 epitopes are highlighted in blue (IgE) and red (IgG). p values were calculated by Mann-Whitney test. (B) Antibody binding Z score for a representative Ara h 7 epitope before (week 0) and after OIT (week 52). (Left) IgE Z scores are shown; (right) IgG Z scores are shown. Critical residues in the dominant footprint are highlighted in red. Each point represents one patient. Statistical significance was determined by paired t test. (C) Change in IgE (blue) and IgG (red) binding Z score to pre-existing epitopes after OIT. Each bar represents the mean log2 fold change of Z scores for all pre-existing epitope peptides (Z score > 3.5 at week 0) at week 52 versus at week 0 for the OIT subject indicated by the x axis. Error bars denote SEM. (D–F) Peptide binding Z scores changes of representative patients exhibiting both IgE decrease and IgG increase (D), IgE unchanged but IgG increase (E), and mixed changes in IgE and IgG (F). Only peptides with either week 0 or week 52 reactivity were examined. Each dot represents one peptide. Statistical significance was determined by Wilcoxon signed rank test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant.

Figure 6

IgE and IgG antibody footprints for the same peanut epitopes are highly similar within individuals (A–C) Representative high-resolution antibody footprints as in Figures 2A–2C, comparing week 0 IgE (top row) and week 52 IgG (bottom row) from the same patient (B04) for peptides Ara h 2.02 aa21–40 (A), Ara h 3.01 aa301–320 (B), and Ara h 7.01 aa71–90 (C). (D) Pearson correlation coefficient between week 0 IgE and week 52 IgG footprints from the same patient for the peanut peptide indicated by the y axis. Each dot represents an IgE-IgG correlation coefficient for one patient. Red bar denotes the mean IgE-IgG correlation of all patients who are reactive to the given peptide.