Phage Immunoprecipitation Sequencing Research Articles

Phenotypic and oncological insights in ANNA1 autoimmunity: Age stratification and biomarker analysis.

To describe the phenotypes, oncological associations, biomarker profiles, and outcomes across different age groups in patients with ANNA1 (anti-Hu) autoimmunity. A retrospective review of patients with ANNA1-IgG in serum/CSF between January 1, 2001, and December 31,2019 was performed. Patients were classified into three groups based on the age of symptom onset. Phage immunoprecipitation sequencing (PhIP-Seq) and neurofilament light chain (NfL) measurements were done in patient sera/CSF with archived samples. Of 122 patients, 81 (66%), 20 (16%), and 21 (17%) patients belonged to older adults, young adults, and pediatric groups, respectively. Lung cancer and neuromuscular presentations were more common in older adults (p < 0.001), while limbic encephalitis and neuroblastoma were more common in pediatric patients (p < 0.005). Most young adults (75%) did not have cancer identified. Proportions of patients with a favorable response to immunotherapy were 20%, 30%, and 52% among older adults, young adults, and pediatric groups, respectively. PhIP-Seq demonstrated significant enrichment for ELAVL4 peptides especially for amino acids 240-289, in the majority of samples evaluated (36/67, 54%). ZIC and SOX2 peptides were significantly enriched in those with central nervous system presentations. Serum NfL levels were elevated in patients with cancer and those with poor long-term outcomes. Young adults with ANNA1 autoimmunity phenotypically resembled older adults but rarely had an underlying cancer. Pediatric patients frequently presented with limbic encephalitis and neuroblastoma and often responded favorably to immunotherapy. Distinct antigenic signatures may underlie differences in clinical presentations. Serum NfL levels may be a biomarker of poor long-term outcomes in ANNA1 autoimmunity.

Ocular Mucous Membrane Pemphigoid Demonstrates a Distinct Autoantibody Profile from Those of Other Autoimmune Blistering Diseases: A Preliminary Study.

Background: Ocular predominant mucous membrane pemphigoid (oMMP) is a severe subtype of autoimmune blistering disease (AIBD), which can result in scarring and vision loss. The diagnosis of oMMP is challenging as patients often have undetectable levels of circulating autoantibodies by conventional assays. Likewise, the principal autoantigen in oMMP has been an area of debate. Methods: In this preliminary experiment, we performed Phage Immunoprecipitation Sequencing (PhIP-seq) on sera from patients with oMMP, as well as non-ocular MMP, bullous pemphigoid, and mucocutaneous-type pemphigus vulgaris. Results: We identified several autoantigens unique to oMMP relative to other AIBDs. We then cross-referenced these antigens against previously published single-nuclei datasets, as well as the International Mouse Phenotyping Consortium Database. Several protein hits identified in our study demonstrated enriched expression on the anterior surface epithelia, including TNKS1BP1, SEC16B, FNBP4, CASZ1, GOLGB1, DOT1L, PRDM 15, LARP4B, and RPL6. Likewise, a previous study of mouse knockout models of murine analogs CASZ1, HIP1, and ELOA2 reported that these mice showed abnormalities in terms of the ocular surface and development in the eyes. Notably, PhIP-seq failed to identify the canonical markers of AIBDs such as BP180, BP230, desmogleins 1 and 3, or integrin β4, indicating that the patient autoantibodies react with conformational epitopes rather than linear epitopes. Conclusions: oMMP patients demonstrate a unique autoantibody repertoire relative to the other AIBDs. Further validation of the identified autoantibodies will shed light on their potentially pathogenic role.

Comprehensive Enteroviral Serology Links Infection and Anti-Melanoma Differentiation-Associated Protein 5 Dermatomyositis.

Idiopathic inflammatory myopathies (IIMs) are a group of heterogeneous, systemic autoimmune diseases characterized by specific clinical features and, frequently, skeletal muscle inflammation. Specific subtypes of IIMs can be characterized by myositis-specific autoantibodies and are associated with distinct clinical phenotypes. Here, we focus on anti-melanoma differentiation-associated protein 5 (MDA5)-positive myositis and anti-signal recognition particle (SRP)-positive myositis, both of which exhibit seasonality but lack known environmental triggers. We employed Phage ImmunoPrecipitation Sequencing to profile serum antibodies against the human proteome, the human virome, and a comprehensive enterovirus library. We analyzed sera from 57 patients with anti-MDA5 autoantibodies and 57 patients with anti-SRP autoantibodies, as well as 57 healthy controls. All groups were matched for age, sex, and race. Our autoantibody profiling results define specific immunogenic regions within the MDA5 and SRP autoantigens. We also discovered that in MDA5 sera, versus SRP sera, there was an elevated antibody response to the viral capsid protein 1 (VP1) of enterovirus B, which was accompanied by a decreased antibody response to rhinovirus A. Considering the role of MDA5 as a sensor of picornaviral infections and a mediator of inflammatory signaling, our data suggest a novel etiologic link between enterovirus infection and anti-MDA5 dermatomyositis.

Anti-peptide antibodies, anti-SNRK and anti-HUWE1 antibodies, as potential predictors of good response to tofacitinib therapy in rheumatoid arthritis patients.

To maximize the cost-effectiveness of tofacitinib, one of Janus kinase inhibitors, there is an unmet need to identify predictors of therapeutic response. Utilizing phage immunoprecipitation sequencing (PhIP-Seq), we aim to identify peptide biomarkers for predicting good response to tofacitinib in rheumatoid arthritis (RA) patients. We enrolled 106 patients who had received 24-week tofacitinib therapy, including twelve patients undergoing PhIP-Seq analysis in the discovery stage and ninety-four patients validated with enzyme-linked immunosorbent assay (ELISA) in the replication stage. Disease activity was assessed using the 28-joint disease activity score-erythrocyte sedimentation rate, and therapeutic response was evaluated using EULAR response criteria. Plasma levels of caspase-1 and IL-18 were determined using ELISA. PhIP-Seq analysis identified antibodies to sucrose non-fermenting-related kinase (SNRK) and HUWE1 (ubiquitin E3 ligase) as peptide biomarkers for discriminating good responders from the non-good responders. Using ELISA for validation on another cohort, an optimal cut-off value of anti-SNRK antibody for predicting good response was 0.381, with AUC 0.823, specificity 80.6%, and sensitivity 78.1% (p = 3.01E-07), and anti-HUWE1 antibody at 0.362, with AUC 0.740, specificity 74.2%, and sensitivity 62.5% (p < 0.001). Plasma levels of anti-SNRK and anti-HUWE1 antibodies were positively correlated with levels of caspase-1 and IL-18 (both p < 0.05). Multivariate logistic regression analysis revealed anti-SNRK antibody as a significant predictor of good therapeutic response. After tofacitinib therapy, anti-SNRK antibody levels significantly declined in good responders, but not in non-good responders. We identify two peptide antibodies, anti-SNRK and anti-HUWE1 antibodies, as pretreatment predictors of good therapeutic response to tofacitinib in RA patients.

Comprehensive phage display viral antibody profiling using VirScan: potential applications in chronic immune-mediated disease.

Phage immunoprecipitation sequencing (PhIP-Seq) is a high-throughput platform that uses programmable phage display for serology. VirScan, a specific PhIP-Seq library encoding viral peptides from all known human viruses, enables comprehensive quantification of past viral exposures. We review its use in immune-mediated diseases (IMDs), highlighting its utility in identifying viral exposures in the context of IMD development. Finally, we evaluate its potential for precision medicine by integrating it with other large-scale omics data sets.

PhIP-Seq: methods, applications and challenges.

Phage-immunoprecipitation sequencing (PhIP-Seq) technology is an innovative, high-throughput antibody detection method. It enables comprehensive analysis of individual antibody profiles. This technology shows great potential, particularly in exploring disease mechanisms and immune responses. Currently, PhIP-Seq has been successfully applied in various fields, such as the exploration of biomarkers for autoimmune diseases, vaccine development, and allergen detection. A variety of bioinformatics tools have facilitated the development of this process. However, PhIP-Seq technology still faces many challenges and has room for improvement. Here, we review the methods, applications, and challenges of PhIP-Seq and discuss its future directions in immunological research and clinical applications. With continuous progress and optimization, PhIP-Seq is expected to play an even more important role in future biomedical research, providing new ideas and methods for disease prevention, diagnosis, and treatment.

Clinical application of phage immunoprecipitation sequencing to diagnose enterovirus D68 as the underlying etiology in a case of Gullain-Barré syndrome.

Gullain-Barré syndrome (GBS) is an acute peripheral neuropathy often preceded by respiratory or gastrointestinal infections, though molecular testing of cerebrospinal fluid (CSF) is often inconclusive. In a recent case of severe pediatric GBS in British Columbia, Canada, we detected CSF antibodies against enterovirus D (EV-D) to link GBS with prior EV-D68 respiratory infection.

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms of myocardial injury in SLE remain poorly understood. In this study, we engineered human cardiac tissues and cultured them with IgG from patients with SLE, with and without myocardial involvement. IgG from patients with elevated myocardial inflammation exhibited increased binding to apoptotic cells within cardiac tissues subjected to stress, whereas IgG from patients with systolic dysfunction exhibited enhanced binding to the surface of live cardiomyocytes. Functional assays and RNA sequencing revealed that, in the absence of immune cells, IgG from patients with systolic dysfunction altered cellular composition, respiration and calcium handling. Phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. Coupling IgG profiling with cell surfaceome analysis identified four potential pathogenic autoantibodies that may directly affect the myocardium. Overall, these insights may improve patient risk stratification and inform the development of new therapeutic strategies.

Phage Immunoprecipitation-Sequencing Reveals CDHR5 Autoantibodies in Select Patients With Interstitial Lung Disease.

Interstitial lung diseases (ILDs) are a heterogeneous group of disorders that can develop in patients with connective tissue diseases. Establishing autoimmunity in ILD impacts prognosis and treatment. Patients with ILD are screened for autoimmunity by measuring antinuclear autoantibodies, rheumatoid factors, and other nonspecific tests. However, this approach may miss autoimmunity that manifests as autoantibodies to tissue antigens not previously defined in ILD. We use Phage Immunoprecipitation-Sequencing (PhIP-Seq) to conduct an autoantibody discovery screen of patients with ILD and controls. We screened for novel autoantigen candidates using PhIP-Seq. We next developed a radio-labeled binding assay and validated the leading candidate in 398 patients with ILD recruited from two academic medical centers and 138 blood bank individuals that formed our reference cohort. PhIP-Seq identified 17 novel autoreactive targets, and machine learning classifiers derived from these targets discriminated ILD serum from controls. Among the 17 candidates, we validated CDHR5 and found CDHR5 autoantibodies in patients with rheumatologic disorders and importantly, patients not previously diagnosed with autoimmunity. Using survival and transplant free-survival data available from one of the two centers, patients with CDHR5 autoantibodies showed worse survival compared with other patients with connective tissue disease ILD. We used PhIP-Seq to define a novel CDHR5 autoantibody in a subset of select patients with ILD. Our data complement a recent study showing polymorphisms in the CDHR5-IRF7 gene locus strongly associated with titer of anticentromere antibodies in systemic sclerosis, creating a growing body of evidence suggesting a link between CDHR5 and autoimmunity.

Efficient encoding of large antigenic spaces by epitope prioritization with Dolphyn

We investigate a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To cover large antigenic spaces, we develop Dolphyn, a method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn compresses the size of a peptide library by 78% compared to traditional tiling, increasing the antibody-reactive peptides from 10% to 31%. We find that the immune system develops antibodies to human gut bacteria-infecting viruses, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis.

Pan-viral serology uncovers distinct virome patterns as risk predictors of hepatocellular carcinoma and intrahepatic cholangiocarcinoma

This study evaluates the pan-serological profiles of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) compared to several diseased and non-diseased control populations to identify risk factors and biomarkers of liver cancer. We used phage immunoprecipitation sequencing, an anti-viral antibody screening method using a synthetic-phage-displayed human virome epitope library, to screen patient serum samples for exposure to over 1,280 strains of pathogenic and non-pathogenic viruses. Using machine learning methods to develop an HCC or iCCA viral score, we discovered that both viral scores were positively associated with several liver function markers in two separate at-risk populations independent of viral hepatitis status. The HCC score predicted all-cause mortality over 8 years in patients with chronic liver disease at risk of HCC, while the viral hepatitis status was not predictive of survival. These results suggest that non-hepatitis viral infections may contribute to HCC and iCCA development and could be biomarkers in at-risk populations.

Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.

Bats are important providers of ecosystem services such as pollination, seed dispersal, and insect control but also act as natural reservoirs for virulent zoonotic viruses. Bats host multiple viruses that cause life-threatening pathology in other animals and humans but, themselves, experience limited pathological disease from infection. Despite bats' importance as reservoirs for several zoonotic viruses, we know little about the broader viral diversity that they host. Bat virus surveillance efforts are challenged by difficulties of field capture and the limited scope of targeted PCR- or ELISA-based molecular and serological detection. Additionally, virus shedding is often transient, thus also limiting insights gained from nucleic acid testing of field specimens. Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a broad serological tool used previously to comprehensively profile viral exposure history in humans, offers an exciting prospect for viral surveillance efforts in wildlife, including bats. Here, for the first time, we apply PhIP-Seq technology to bat serum, using a viral peptide library originally designed to simultaneously assay exposures to the entire human virome. Using VirScan, we identified past exposures to 57 viral genera-including betacoronaviruses, henipaviruses, lyssaviruses, and filoviruses-in semi-captive Pteropus alecto and to nine viral genera in captive Eonycteris spelaea. Consistent with results from humans, we find that both total peptide hits (the number of enriched viral peptides in our library) and the corresponding number of inferred past virus exposures in bat hosts were correlated with poor bat body condition scores and increased with age. High and low body condition scores were associated with either seropositive or seronegative status for different viruses, though in general, virus-specific age-seroprevalence curves defied assumptions of lifelong immunizing infection, suggesting that many bat viruses may circulate via complex transmission dynamics. Overall, our work emphasizes the utility of applying biomedical tools, like PhIP-Seq, first developed for humans to viral surveillance efforts in wildlife, while highlighting opportunities for taxon-specific improvements.

Identification of SKOR2 IgG as a novel biomarker of paraneoplastic neurologic syndrome.

The development of new autoantigen discovery techniques, like programmable phage immunoprecipitation sequencing (PhIP-Seq), has accelerated the discovery of neural-specific autoantibodies. Herein, we report the identification of a novel biomarker for paraneoplastic neurologic syndrome (PNS), Sloan-Kettering-Virus-Family-Transcriptional-Corepressor-2 (SKOR2)-IgG, utilizing PhIP-Seq. We have also performed a thorough clinical validation using normal, healthy, and disease/cancer control samples. Stored samples with unclassified staining at the junction of the Purkinje cell and the granule cell layers were analyzed by PhIP-Seq for putative autoantigen identification. The autoantigen was confirmed by recombinant antigen-expressing cell-based assay (CBA), Western blotting, and tissue immunofluorescence assay colocalization. PhIP-Seq data revealed SKOR2 as the candidate autoantigen. The target antigen was confirmed by a recombinant SKOR-2-expressing, and cell lysate Western blot. Furthermore, IgG from both patient samples colocalized with a commercial SKOR2-specific IgG on cryosections of the mouse brain. Both SKOR2 IgG-positive patients had central nervous system involvement, one presenting with encephalitis and seizures (Patient 1) and the other with cognitive dysfunction, spastic ataxia, dysarthria, dysphagia, and pseudobulbar affect (Patient 2). They had a refractory progressive course and were diagnosed with adenocarcinoma (Patient 1: lung, Patient 2: gallbladder). Sera from adenocarcinoma patients without PNS (n=30) tested for SKOR2-IgG were negative. SKOR2 IgG represents a novel biomarker for PNS associated with adenocarcinoma. Identification of additional SKOR2 IgG-positive cases will help categorize the associated neurological phenotype and the risk of underlying malignancy.

Abstract CT123: Trial in progress: First-in-human immunotherapy-trio for advanced head and neck squamous cell carcinoma

Abstract Background: Glucocorticoid-Induced Tumor Necrosis Factor Receptor-related protein (GITR) is a co-stimulatory pathway that when triggered has potent effects on T-cell memory, proliferation and anti-tumor activity. Preclinical models identified significant synergy between anti-GITR agonist therapy and cancer vaccines to generate stronger tumor specific CD8 T cell responses. DPV-001 is an “off-the-shelf” multivalent autophagosome vaccine generated by in vitro manipulation of the autophagy pathway in human cancer cell lines. The vaccine delivers short-lived proteins (SLiPs) and defective ribosomal products (DRiPs) which are likely the dominant epitopes directly presented by MHC class I of tumor cells; but because of proteosomal degradation, are normally unavailable for cross-presentation, hence the delivery via vaccine. We hypothesize that addition of aGITR to DPV-001 vaccine will augment expansion of reactive CD4 and CD8 T cells, attenuate contraction of this response, and improve the therapeutic effect of treatment, and will result in the development of a coordinated T and B cell response to some of the same proteins, detectable using a cutting-edge seromics approach, as a window to TCR target identification for immunodynamic tracking of induced anti-cancer responses at an advanced level. Methods: Patient recruitment began in August 2022, for this first-in-human immunotherapy-trio study of DPV-001, with sequenced checkpoint inhibition (aPD-1 mAb; retifanlimab), with or without aGITR agonist mAb (INCAGN-1949), in recurrent or metastatic HNSCC (NCT04470024). Patient population to include HPV-positive or HPV-negative, ECOG 0-2, with therapy continued until confirmed progression (RECIST 1.1), up to 2 years. Primary objective is safety, DLT ≤ 33%, with secondary efficacy objectives of ORR (PR+CR) and 2 year OS. Initial safety lead-in (n = 3+3 per arm), will be followed by phase Ib expansion of one/both arms if immunologically promising, 28 patients per arm, futility if &amp;lt;4/15 responses. Study Drugs Cyclophosphamide 300mg/m2 IV, priming Day (-2) onlyVaccine (DPV-001)- Day 1 intranodal US bilateral inguinal- Days 8,15 intradermal, then q2wks to week 22- Thereafter q4wks until progression, up to 2 yearsaPD-1 (retifanlimab) 500mg IV q4wks, start Day 15 (Arms 1 &amp; 2)aGITR (INCAGN01876) 300mg IV q2wks, start Day 1 (Arm 2 only) Response (RECIST 1.1) CT weeks 8 and 12, then q3mos Immunologic Monitoring PBL and sera are collected regularly and PBL are evaluated by flow cytometry. Biopsies obtained at baseline, Day 15 and Day 57, analyzed by mIF and 10x scRNA-Seq. Sera analyzed by phage immunoprecipitation (PhIP) sequencing for reactivity against the human proteome. Immune monitoring modifications that allow for improved characterization of immune cell subsets will be presented. Citation Format: Marcus A. Couey, Matthew Taylor, Tarsem Moudgil, Yoshinobu Koguchi, Anne Stadum, Tanisha Christie, William L. Redmond, Christopher Paustian, Ryan Meng, Venkatesh Rajamanickam, Lessli Rushforth, Abigail Peterson, Brady Bernard, Richard B. Bell, Carlo B. Bifulco, Traci L. Hilton, Shawn M. Jensen, Hong-Ming Hu, Brian Piening, Walter J. Urba, Bernard A. Fox, Rom S. Leidner. Trial in progress: First-in-human immunotherapy-trio for advanced head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT123.

Abstract 5767: Pan-viral response in African Americans and European Americans with hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is often diagnosed at late-stages, with African Americans (AA) being diagnosed at a later stage with worse prognosis and high mortality rates. The poor screening and diagnosis of HCC and differences between racial/ethnic groups show a disproportion in mortality rates. Examining an individual’s viral exposure history might serve as a useful biomarker of early disease that could address and, potentially, alleviate this disparity. We aim to investigate whether viral features can be used to identify HCC and individuals at high risk (HR) of HCC. Additionally, we examine the racial/ethnic differences in viral exposures and whether viral exposure history accounts for epidemiological disparity described previously. Herein we use a technique called VirScan, which conjugates Phage Immunoprecipitation sequencing (PhIP-seq) and DNA sequencing, to profile an individual’s epitope antibody repertoire upon viral exposure. We performed VirScan on serum and plasma samples from 199 AA (130 HR and 79 HCC) and 260 European Americans (EA) (210 HR and 50 HCC) participants from the NCI-UMD cohort (NCT00913757; clinicaltrials.gov). We used a machine-learning XGBoost model to classify viral features that can discriminate HCC or HR. A composite viral score was generated using the SHAP value of the most significant viral features from the model with a higher score more predictive of HCC. Secondary analyses using Wilcoxon-Rank sums tests and Cox proportional hazard analysis revealed that while race/ethnicity did not differentially predict HCC in this cohort, the viral score was significantly higher in AA compared to EA in the HR group (p = 0.027). XGBoost search identified 235 important viral features which distinguish HCC from HR. Out of the 235 viral features, 20 viruses were different between AA and EA (FDR &amp;lt; 0.05). We also found the viral score was positively associated with incident mortality in HCC group over the study period of 8 years (p=0.03). Thus, we demonstrate that the viral history of an individual might be useful in early detection of HCC. This approach could be used to account for the disparities we see in both incidence and mortality of HCC between AA and EA. Citation Format: Theressa Ewa, Whitney L. Do, Limin Wang, Marshonna Forgues, Xin W. Wang. Pan-viral response in African Americans and European Americans with hepatocellular carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5767.

Caveolae-Associated Protein (cavin)-4 Autoantibodies in Immune Mediated Rippling Muscle Disease

Objective To describe a novel autoantibody biomarker of Immune mediated rippling muscle disease (iRMD). Background iRMD is a rare immunotherapy-responsive myopathy characterized by wave-like muscle contractions (rippling) and percussion/stretch-induced muscle mounding. However, serological biomarker of this disease is lacking. Design/Methods A Retrospective review was done to identify iRMD patients with stored sera in Mayo Neuroimmunology laboratory. Archived sera from IRMD patients were evaluated for a common biomarker of IRMD using phage immunoprecipitation sequencing (PhIP-Seq). Results Archival sera from 10 patients with clinical diagnosis of iRMD were retrieved. Whole human proteome PhIP-Seq identified peptides corresponding to different regions of the cavin-4 in sera of iRMD patients. Eight of the ten iRMD cases were positive for cavin-4 IgG by immunofluorescent cell-based-assay (CBA) using cavin-4-transfected COS7 cells. The cavin-4-reactive IgG in all 8 positive sera was of IgG1 subclass. None of the disease control sera (98 immune-mediated myopathy/neuromuscular junction disorders, 20 autoimmune CNS diseases and 123 healthy subjects) contained cavin-4-reactive IgG. Furthermore, none of the iRMD patients' sera were positive for caveolin-3 IgG. The majority of seropositive cases were males (6/8, 75%) with median age of 51 years (range 18-76). Three seropositive patients had co-existing myasthenia gravis (38%). Creatine Kinase was elevated in 6/7 tested patients (median 771 U/L, range: 132-2625 U/L). Muscle biopsy was performed in 7 of the 8 cavin-4 IgG seropositive patients; 6/6 specimens analyzed immunohistochemically revealed a mosaic pattern of sarcolemmal cavin-4 immunoreactivity. Three of 6 seropositive patients who received immunotherapy had complete resolution of symptoms; one had mild improvement and two had no change. Conclusions Cavin-4 IgG is a novel and specific serological autoantibody biomarker identified in iRMD. Depletion of cavin-4 expression in iRMD patient muscle biopsies suggests the potential role of this autoantigen in disease pathogenesis.

Comprehensive profiling of antibody responses to the human anellome using programmable phage display.

Anelloviruses represent a major constituent of the commensal human virome; however, little is known about their immunobiology. Here, we present "AnelloScan," a T7 phage library representing the open reading frame 1 (ORF1), ORF2, ORF3, and torque teno virus (TTV)-derived apoptosis-inducing protein (TAIP) sequences of more than 800 human anelloviruses and profile the antibody reactivities of serum samples from a cross-sectional cohort of 156 subjects by using phage-immunoprecipitation sequencing (PhIP-Seq). A majority of anellovirus peptides are not reactive in any of the subjects tested (n= ∼28,000; ∼85% of the library). Antibody-reactive peptides are largely restricted to the C-terminal region of the capsid protein ORF1. Moreover, using a longitudinal cohort of matched blood-transfusion donors and recipients, we find that most transmitted anelloviruses do not elicit a detectable antibody reactivity in the recipient and that the remainder elicit delayed responses appearing ∼100-150days after transfusion.

BIPS-A code base for designing and coding of a Phage ImmunoPrecipitation Oligo Library.

BIPS (Build Phage ImmunoPrecipitation Sequencing library) is a software that converts a list of proteins into a custom DNA oligonucleotide library for the PhIP-Seq system. The tool creates constant-length oligonucleotides with internal barcodes, while maintaining the original length of the peptide. This allows using large libraries, of hundreds of thousands of oligonucleotides, while saving on the costs of sequencing and maintaining the accuracy of oligonucleotide reads identification. BIPS is available under GNU public license from: https://github.com/kalkairis/BuildPhIPSeqLibrary.

Detection of Antinuclear Antibodies Targeting Intracellular Signal Transduction, Metabolism, Apoptotic Processes and Cell Death in Critical COVID-19 Patients.

The heterogeneity of the coronavirus disease of 2019 (COVID-19) lies within its diverse symptoms and severity, ranging from mild to lethal. Acute respiratory distress syndrome (ARDS) is a leading cause of mortality in COVID-19 patients, characterized by a hyper cytokine storm. Autoimmunity is proposed to occur as a result of COVID-19, given the high similarity of the immune responses observed in COVID-19 and autoimmune diseases. Here, we investigate the level of autoimmune antibodies in COVID-19 patients with different severities. Initial screening for antinuclear antibodies (ANA) IgG using ELISA revealed that 1.58% (2/126) and 4% (5/126) of intensive care unit (ICU) COVID-19 cases expressed strong and moderate ANA levels, respectively. An additional sample was positive with immunofluorescence assays (IFA) screening. However, all the non-ICU cases (n=273) were ANA negative using both assays. Samples positive for ANA were further confirmed with large-scale autoantibody screening by phage immunoprecipitation-sequencing (PhIP-Seq). The majority of the ANA-positive samples showed "speckled" ANA pattern by microscopy and revealed autoantibody specificities that targeted proteins involved in intracellular signal transduction, metabolism, apoptotic processes, and cell death by PhIP-Seq; further denoting reactivity to nuclear and cytoplasmic antigens. Our results further support the notion of routine screening for autoimmune responses in COVID-19 patients, which might help improve disease prognosis and patient management. Further, results provide compelling evidence that ANA-positive individuals should be excluded from being donors for convalescent plasma therapy in the context of COVID-19.

Autoantibody 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.