title: High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium tuberculosis Infections.
abstract: The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens.
authors.affiliation: Doctoral Program in the Chemical and Biological Sciences The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.|Opko Health, Inc. 5353 Parkside Drive Jupiter, Florida 33458.
authors.forename: Kimberly R
authors.initials: KR
authors.lastname: Mendes
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.|Doctoral Program in the Chemical and Biological Sciences The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Marie Lynne
authors.initials: ML
authors.lastname: Malone
authors.affiliation: Opko Health, Inc. 5353 Parkside Drive Jupiter, Florida 33458.
authors.forename: John Maina
authors.initials: JM
authors.lastname: Ndungu
authors.affiliation: Opko Health, Inc. 5353 Parkside Drive Jupiter, Florida 33458.
authors.forename: Irena
authors.initials: I
authors.lastname: Suponitsky-Kroyter
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Valerie J
authors.initials: VJ
authors.lastname: Cavett
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: McEnaney
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.|Doctoral Program in the Chemical and Biological Sciences The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: MacConnell
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Todd M
authors.initials: TM
authors.lastname: Doran
authors.affiliation: Department of Science and Technology/National Research Foundation Centre of Excellence for Biomedical TB Research/Medical Research Council Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: Katharina
authors.initials: K
authors.lastname: Ronacher
authors.affiliation: Department of Science and Technology/National Research Foundation Centre of Excellence for Biomedical TB Research/Medical Research Council Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: Kim
authors.initials: K
authors.lastname: Stanley
authors.affiliation: Opko Health, Inc. 5353 Parkside Drive Jupiter, Florida 33458.
authors.forename: Ofelia
authors.initials: O
authors.lastname: Utset
authors.affiliation: Department of Science and Technology/National Research Foundation Centre of Excellence for Biomedical TB Research/Medical Research Council Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Walzl
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Paegel
authors.affiliation: Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter, FL 33458.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kodadek
mesh_terms: D000217:Acyltransferases / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D057166:High-Throughput Screening Assays; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D055985:Latent Tuberculosis / Q000097:blood / Q000276:immunology* / Q000382:microbiology; D008024:Ligands; D009169:Mycobacterium tuberculosis; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology*; D019151:Peptide Library; D060327:Solid-Phase Synthesis Techniques; D013329:Structure-Activity Relationship; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000276:immunology* / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D007074:Immunoglobulin G; D008024:Ligands; D009842:Oligopeptides; D019151:Peptide Library; D004247:DNA; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis