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Reading the Antibody Repertoire Against Viruses, Microbes, and the Self

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Reading the Antibody Repertoire Against Viruses, Microbes, and the Self Old Herborn University Seminar Monograph 31: Evolutionary biology of the virome, and impacts in human health and disease. Editors: Peter J. Heidt, Pearay L. Ogra, Mark S. Riddle and Volker Rusch. Old Herborn University Foundation, Herborn, Germany: 89-96 (2017). READING THE ANTIBODY REPERTOIRE AGAINST VIRUSES, MICROBES, AND THE SELF URI LASERSON Department of Genetics and Genomic Sciences and Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA SUMMARY Accessing the information in the antibody repertoire promises to provide an unprecedented window into the exposure history and disease susceptibility of an individual. However, the non-heritability and massive diversity of the receptors are major hurdles to interpreting the repertoire. We have devel- oped the phage immunoprecipitation sequencing assay (PhIP-seq) that inex- pensively generates high-dimensional functional profiles of a complex mix- ture of antibodies of unknown specificity. The assay leverages the recent ad- vances in next-generation DNA sequencing along with synthesis of large pools of DNA oligonucleotides. We have successfully applied PhIP-seq to characterize auto-antigen profiles in patients with autoimmune diseases along with a global survey of antiviral immune responses. We are further developing the assay to apply to host-microbiome interactions, among other application areas. INTRODUCTION The repertoire of antibodies present in studying antibody and T cell receptor a single drop of blood should reveal an (TCR) repertoires (Georgiou et al., enormous amount of information about 2014). The use of NGS to study the im- an individual, such as their history of mune repertoire has resulted in many infection, travel, vaccines, allergies, insights about the core function of im- and even their susceptibility to future munity and its role in virtually every diseases (Lerner et al., 1991). How- disease, including infections (Parames- ever, multiple hurdles have obstructed waran et al., 2013), cancer (Li et al., our ability to glean such insights from 2016; Liu and Mardis, 2017), and the repertoire: proteins are more diffi- autoimmunity (Dekosky et al., 2015). cult to work with than nucleic acids, A holy grail in the field of adaptive the non-germline nature of the reper- immunity is the creation of a mapping toire complicates population studies between antibody/TCR sequences and (since most antibodies are rare antibod- their cognate epitopes, such that an ies), and the enormous sequence diver- antigen/epitope can be determined sity has historically exceeded the capa- purely from inspecting the antibody bilities of any measurement technol- sequence and vice versa. But despite ogy. The introduction of next-genera- the massive increase in antibody se- tion DNA sequencing technology quence data (Breden et al., 2017), most (NGS) a decade ago (Shendure et al., studies typically report either aggregate 2017) has reinvigorated excitement in statistics (e.g., repertoire diversity met- 89 rics, germline gene usage) or compare as random oligomers with lengths <20 them across experimental conditions to amino acids. However, libraries of ran- discover biomarkers (Yaari and Klein- dom peptides "waste" clones on se- stein, 2015). Alternatively, some stud- quences that are not observed in nature. ies have characterized antibody reper- Furthermore, the use of short peptides toires in the context of a single antigen precludes their ability to exhibit natural and require low-throughput sorting of structure. To circumvent these limita- antigen-specific cells. The ability to tions, we have leveraged improvements predict immune receptor function from in the ability to synthesize large, com- its sequence has only been demon- plex pools of DNA oligonucleotides strated in very narrow situations and (Kosuri and Church, 2014). Similarly primarily for TCRs (Emerson et al., to pooled-screen experiments, the abil- 2017; Glanville et al., 2017). ity to synthesize large pools of DNA Here we describe phage immuno- oligos to-order allows us to "synthe- precipitation sequencing (PhIP-seq) size" many experiments and execute (Larman et al., 2011), an NGS-based them in parallel (Gasperini et al., assay that can determine the specificity 2016). of a repertoire of antibodies against As described below, PhIP-seq has hundreds of thousands of antigens been used successfully to generate simultaneously. The assay makes use high-dimensional profiles of antibody of phage display libraries containing a immunity in the context of autoimmun- set of antigens, each of which will be ity and viral infection. In the following measured for binding to some antibody sections, we review some of the results of interest. Because the universe of of these efforts, along with preliminary possible antigens grows exponentially methods for expanding the scope of with peptide length, such peptide li- PhIP-seq to bacterial antigens. braries have typically been constructed PHAGE IMMUNOPRECIPITATION SEQUENCING Given a monoclonal antibody or a com- limited by what can be cloned into the plex mixture of antibodies (e.g., serum) phage display library. of unknown specificity, the PhIP-seq For example, the set of human assay can determine the specificity of protein sequences can be tiled with a the antibodies from a large set of candi- set of ~400,000 unique peptide 36- date epitopes that have been selected in mers. The DNA oligos that code for the advance. Specifically, the "query anti- 36-mers can be synthesized by a body" is used to immunoprecipitate a commercial DNA vendor (e.g., Twist phage display library expressing many Bioscience or Agilent Technologies) antigens of interest. The phage clones and cloned into a commercially availa- that are pulled down by the antibody ble bacteriophage T7 protein display are quantified using next-generation system (e.g., Novagen T7Select) to DNA sequencing. Therefore, in a yield a phage library containing every single ~$30 assay, small amounts of possible human protein 36-mer. The antibody (nanogram to microgram phage library can then be used to quantities) can be assayed against hun- identify unknown auto-antigens using dreds of thousands of candidate serum samples from patients with epitopes in parallel. In principle, the autoimmune diseases. diversity of the epitope library is only It is estimated that a majority of 90 antibody epitopes are "conformational" translational modifications when appli- rather than linear (Sela et al., 1967; cable. These limitations ultimately lead Barlow et al., 1986). A clear limitation to a loss of sensitivity and make PhIP- of this method is that the epitopes are seq somewhat unsuitable for diagnostic limited to the length of DNA oligo- applications. nucleotides that can be synthesized en It is our experience that robust im- masse. As of this writing, synthesis of mune responses generally include at large oligo pools is generally limited to least some responses to linear epitopes. 200-300 bp sequences, in which some Therefore, PhIP-seq is highly suitable of the sequence must be used for com- for hypothesis generation experiments, mon adaptor sequence for cloning similarly to genome wide association (allowing ~56 aa peptides). Even if we studies (GWAS). Instead of testing could synthesize or assemble sig- SNPs for association with a phenotype, nificantly larger pieces of DNA, we we can use PhIP-seq to test whether would eventually reach the capacity of particular antibody specificities are the phage display system to display associated with a phenotype. Despite larger proteins without interfering with its limitations, PhIP-seq provides an the assembly of infective phage inexpensive means to generate a very particles. Furthermore, because the high-dimensional characterization of peptides are processed in E. coli, we the antibody specificity repertoire. cannot include any mammalian post- DISCOVERY OF AUTO-ANTIGENS PhIP-seq was first demonstrated by body that is not causing any pathology. building a library of ~400,000 36-mer Interestingly, some auto-antigen speci- peptides tiling >24,000 human open ficities were observed in >20 healthy reading frames with 7 aa overlaps (Lar- control subjects, such at MAGEE1, man et al., 2011). Our initial test ACVR2B, and TTN. But given the searched for auto-antigens in several healthy status of these individuals, the cancer patients with paraneoplastic most likely explanation is that they neurological disorder (PND), a cancer- represent common cross-reactivities. induced autoimmune syndrome. After Using the samples from T1D patients, successfully detecting known and novel we analysed the sensitivity of the PhIP- auto-antigens in these patients, we un- seq assay by comparing peptide enrich- dertook a larger survey of ~300 sub- ments to RIA assays for known auto- jects with multiple sclerosis (MS), type antibodies against insulin, ZnT8A, 1 diabetes (T1D), or rheumatoid arthri- GAD2, and PTPRN. PhIP-seq with the tis (RA), along with healthy controls 36-mer library was only able to recover (Larman et al., 2013A). Analysis of the reactivities against GAD2 and PTPRN repertoire of autoantibodies in healthy in some patients. Analysis of RA pa- controls revealed that most individuals tients did not find any new auto-anti- exhibit antibodies against a variety of gens; however, we were able to cluster self-epitopes. However, the vast major- a subset of patients into one of two ity of these self-specificities were "pri- specificity profiles, which was not cor- vate" in the sense that they were ob- related
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