Antibody epitope repertoire analysis enables rapid antigen discovery and multiplex serology

Abstract

The detection of pathogen-specific antibodies remains a cornerstone of clinical diagnostics. Yet, many test exhibit undesirable performance or are completely lacking. Given this, we developed serum epitope repertoire analysis (SERA), a method to rapidly discover conserved, pathogen-specific antigens and their epitopes, and applied it to develop an assay for Chagas disease caused by the protozoan parasite Trypanosoma cruzi. Antibody binding peptide motifs were identified from 28 Chagas repertoires using a bacterial display random 12-mer peptide library and next-generation sequencing (NGS). Thirty-three motifs were selected and mapped to candidate Chagas antigens. In a blinded validation set (n = 72), 30/30 Chagas were positive, 30/30 non-Chagas were negative, and 1/12 Leishmania sp. was positive. After unblinding, a Leishmania cross-reactive epitope was identified and removed from the panel. The Chagas assay exhibited 100% sensitivity (30/30) and specificity (90/90) in a second blinded validation set including individuals with other parasitic infections. Amongst additional epitope repertoires with unknown Chagas serostatus, assay specificity was 99.8% (998/1000). Thus, the Chagas assay achieved a combined sensitivity and specificity equivalent or superior to diagnostic algorithms that rely on three separate tests to achieve high specificity. NGS-based serology via SERA provides an effective approach to discover antigenic epitopes and develop high performance multiplex serological assays.

Serum Epitope Repertoire Analysis Enables Early Detection of Lyme Disease with Improved Sensitivity in an Expandable Multiplex Format

“…In this manner the beads were washed 5 times before growth media was added and the retained E.coli cells grown overnight. All subsequent steps were identical for IgG and IgM screening as described (16).…”

“…We previously described a next-generation sequencing (NGS) based serology platform enabling discovery of antigenic epitopes, with effectively unlimited multiplexing, termed serum epitope repertoire analysis (SERA) (16). SERA integrates high-diversity random peptide libraries (16), NGS, and bioinformatic tools and computational analytics (17)(18)(19) to discover a multitude of pathogen-specific antibody epitopes and optimize their combinations to create high performance serology tests (16). Application of SERA to specimens from subjects with chronic Chagas disease resulted in an IgG assay exhibiting 100% sensitivity with 99.6% specificity (16), exceeding the performance of current parallel independent tests required to achieve high specificity (20).…”

“…SERA integrates high-diversity random peptide libraries (16), NGS, and bioinformatic tools and computational analytics (17)(18)(19) to discover a multitude of pathogen-specific antibody epitopes and optimize their combinations to create high performance serology tests (16). Application of SERA to specimens from subjects with chronic Chagas disease resulted in an IgG assay exhibiting 100% sensitivity with 99.6% specificity (16), exceeding the performance of current parallel independent tests required to achieve high specificity (20). Because SERA uses a library of ~10 billion random peptides that can effectively mimic nearly any protein-based antigen, the high-content archival datasets that SERA generates can be analyzed for antibodies associated with many different infections (21), allergies, and autoimmune diseases (19,21).…”

“…To utilize the full set of motifs for disease classification, a composite score was calculated by summing the standardized enrichment values for each motif as described (16) on Figure 4A).…”

Autoantibody landscape of advanced prostate cancer

“…Given that metastatic castration-resistant prostate cancer (mCRPC) represents one of the leading causes of cancer-associated death in men, we sought to characterize the autoantibody landscape of this disease. Utilizing SERA, PIWAS, and IMUNE 25,31 , we performed an unbiased analysis of autoantibodies enriched in the serum of mCRPC patients compared to healthy controls. Specifically, we leveraged DNA-sequence level information from the assay to identify not only the proteins but also the specific epitopes (sub-peptides) within the full-length proteins that were putatively antigenic in mCRPC.…”