biostudies-literatureVazquez SEParker Institute for Cancer ImmunotherapyFRMEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNational Institute of Child Health and DevelopmentImagine InstituteNational Institute of Diabetes and Digestive and Kidney DiseasesNational Institute of General Medical SciencesDivision of Intramural Research, National Institute of Allergy and Infectious DiseasesNational Institute of Allergy and Infectious DiseasesJuvenile Diabetes Research Foundation United States of AmericaLaboratory of Human Genetics of Infectious DiseasesNIDDK NIH HHSChan Zuckerberg BiohubHelmsley Charitable TrustAmerican Diabetes AssociationUCSF-CTSI TL1NIGMS NIH HHSe78550https://www.ebi.ac.uk/biostudies/studies/S-EPMC9711525biostudies-literatureUnknown11Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.eLifeAutoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq.PMC97115255T32GM007618MD-PhD program1ZIAAI0011755P01AI118688TR0018711-19-PDF-1311 ZIA AI001222EA201706380201F30DK1239151R61HD105590Quandt ZLiu JProekt IYu DZhang SYTorgerson TRWang CYFerre EMNMiao BShimizu CLandegren NSowa GBacchetta RZorn KAnderson MSDelmonte OMDeRisi JLLynch KBastard PChan AYBurns JCTagi VMWilson MRTremoulet ANotarangelo LDCasanova JLEriksson DKung AFMandel-Brehm CKampe OBodansky ADobbs KLionakis MSMitchell AMann SAVazquez SEfalseAutoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq.Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.2022-01-01T00:00:00Z2022 Oct2025-04-05T09:43:47.061Z2025-04-05T09:43:47.061ZS-EPMC97115253630062310.7554/eLife.78550