REAP: A platform to identify autoantibodies that target the human exoproteome

Abstract

Autoantibodies that recognize extracellular protein epitopes (the "exoproteome") exert potent functional effects that underlie numerous disease processes. Identifying these antibodies can thus provide insights into the pathophysiology of a wide spectrum of illnesses and therapeutic strategies to treat them. Here, we developed Rapid Extracellular Antigen Profiling (REAP) as a technique for comprehensive and high-throughput discovery of exoproteome-targeting autoantibodies. With REAP, patient samples are applied to a genetically-barcoded library containing 2,688 unique members of the human exoproteome displayed on the surface of yeast. Antibody-coated cells are isolated by magnetic selection and deep sequencing of their barcodes is used to identify the displayed antigens, thereby converting an antibody:antigen binding event into a digital sequencing readout. To benchmark the performance of REAP, we screened 77 patients with the rare monogenic autoimmune disease autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). REAP sensitively and specifically detected known autoantibody reactivities in APECED, including responses against type I interferons, IL-17, IL-22, and gastric intrinsic factor. REAP also identified highly prevalent reactivities that had not been previously described such as those against the glycoprotein hormone GPHB5. We additionally screened 106 patients with systemic lupus erythematosus (SLE) and identified novel autoantibody reactivities against a diverse set of antigens including growth factors, extracellular matrix components, cytokines, and immunomodulatory proteins. Several of these responses were associated with disease severity and specific clinical manifestations of SLE, including autoantibodies that target immunoreceptors, antagonize the pro-inflammatory cytokine IL-33, and recognize endosialin (CD248) and the chemokine CCL8. In summary, these findings demonstrate the utility of REAP to atlas the expansive landscape of exoproteome-targeting autoantibodies in patients.

The relationship between autoantibodies targeting GPCRs and the renin-angiotensin system associates with COVID-19 severity

“…Anyhow, the results of our work underscores recent studies 3,6,10,12 that report the generation of autoantibodies following SARS-CoV-2 infection. Importantly, our data indicate that an additional immunological layer is present where autoantibodies targeting GPCRs and RASrelated molecules are associated with COVID-19 burden.…”

“…Anyhow, the results of our work underscores recent studies 3,6,10,12 The Random Forest model revealed the interaction between autoantibodies targeting CXCR3 and AT1R as the most important predictors of COVID-19 severity. There is an essential biological connection between CXCR3 and AT1R.…”

“…High titers of neutralizing immunoglobulin G (IgG) autoantibodies against type I interferons (IFNs) have been reported in patients with life-threatening COVID-19 10 . Most recently, a wide range of autoantibodies in patients with COVID-19 have been characterized using rapid extracellular antigen profiling (REAP) 11 , a technology for comprehensive and high-throughput identification of autoantibodies recognizing 2,770 extracellular and secreted protein components of the exoproteome (extracellular protein epitopes) 12 . Wang, E. Y. et al 11 showed that both healthy controls and COVID-19 patients have multiple autoantibodies against the exoproteome.…”

Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy

“…In APECED patients, the detection of several tissue antigen-directed autoantibodies has been associated with the presence of corresponding organ-specific autoimmune manifestations (13,52,(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75). New experimental approaches such as phage/bacterial peptide display (PhiP-Seq) and yeast surface display (REAP) have been recently used to uncover novel autoantigen specificities such as KHDC3L, associated with primary ovarian failure, RFX6, associated with intestinal dysfunction, and colipase, associated with exocrine pancreatic insufficiency (76,77). However, for most of the tissue antigentargeted autoantibodies detected in APECED patients, it is difficult to reliably predict the development of the corresponding autoimmune manifestation at the individual patient level as their sensitivity and specificity is not very high.…”

Mapping Autoantibodies in Children With Acute Rheumatic Fever

“…Similarly, membrane associated antigens may be misfolded or under-represented on both of the array platforms utilised given the uniform approach required to express and purify such large numbers of antigens in parallel. To overcome this limitation, and expand the antigen space examined in the context of ARF, alternative approaches such as Phage Immunoprecipitation Sequencing [PhiP −Seq (53)] and Rapid Extracellular Antigen Profiling [REAP (54)] of the human proteome could be considered in future studies. Finally, the initial array analysis was based on small patient numbers and it is possible that additional ARF autoantibodies would be detected with larger cohorts.…”