High-throughput discovery of MHC class I- and II-restricted T cell epitopes using synthetic cellular circuits.

Antigen discovery technologies have largely focused on major histocompatibility complex (MHC) class I-restricted human T cell receptors (TCRs), leaving methods for MHC class II-restricted and mouse TCR reactivities relatively undeveloped. Here we present TCR mapping of antigenic peptides (TCR-MAP), an antigen discovery method that uses a synthetic TCR-stimulated circuit in immortalized T cells to activate sortase-mediated tagging of engineered antigen-presenting cells (APCs) expressing processed peptides on MHCs. Live, tagged APCs can be directly purified for deconvolution by sequencing, enabling TCRs with unknown specificity to be queried against barcoded peptide libraries in a pooled screening context. TCR-MAP accurately captures self-reactivities or viral reactivities with high throughput and sensitivity for both MHC class I-restricted and class II-restricted TCRs. We elucidate problematic cross-reactivities of clinical TCRs targeting the cancer/testis melanoma-associated antigen A3 and discover targets of myocarditis-inciting autoreactive T cells in mice. TCR-MAP has the potential to accelerate T cell antigen discovery efforts in the context of cancer, infectious disease and autoimmunity.

Elucidation of E3 ubiquitin ligase specificity through proteome-wide internal degron mapping.

The ubiquitin-proteasome system plays a critical role in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for a few of the 600 E3s. Here, we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a proteome-scale in HEK-293T cells. We employ global protein stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs, of which we uncovered 16 pairs that revealed extensive degron variability and structural determinants. We provide the visualization of these data on the public DegronID data browser as a resource for future exploration.

Parasitic, bacterial, viral, immune-mediated, metabolic and nutritional factors associated with nodding syndrome.

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis, and a hypothetical causal model was constructed using structural equation modelling. Of 607 children with nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio 7.04, 95% confidence interval 2.28-21.7), Necator americanus infection (odds ratio 2.33, 95% confidence interval 1.02-5.3), higher antimalarial seroreactivity (odds ratio 1.75, 95% confidence interval 1.20-2.57), higher vitamin E concentration (odds ratio 1.53 per standard deviation increase, 95% confidence interval 1.07-2.19) and lower vitamin B12 concentration (odds ratio 0.56 per standard deviation increase, 95% confidence interval 0.36-0.87) were associated with higher odds of nodding syndrome. In a structural equation model, we hypothesized that Mansonella perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of nodding syndrome while lower vitamin B12 concentration, Necator americanus and malaria infections resulted from having nodding syndrome. We found no evidence that Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 and other factors were associated with nodding syndrome. Our results argue against several previous causal hypotheses including Onchocerca volvulus. Instead, nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect.

Coexisting autoantibodies against transcription factor Sp4 are associated with decreased cancer risk in patients with dermatomyositis with anti-TIF1γ autoantibodies.

OBJECTIVES: In dermatomyositis (DM), autoantibodies are associated with unique clinical phenotypes. For example, anti-TIF1γ autoantibodies are associated with an increased risk of cancer. The purpose of this study was to discover novel DM autoantibodies. METHODS: Phage ImmunoPrecipitation Sequencing using sera from 43 patients with DM suggested that transcription factor Sp4 is a novel autoantigen; this was confirmed by showing that patient sera immunoprecipitated full-length Sp4 protein. Sera from 371 Johns Hopkins patients with myositis (255 with DM, 28 with antisynthetase syndrome, 40 with immune-mediated necrotising myopathy, 29 with inclusion body myositis and 19 with polymyositis), 80 rheumatological disease controls (25 with Sjogren's syndrome, 25 with systemic lupus erythematosus and 30 with rheumatoid arthritis (RA)) and 200 healthy comparators were screened for anti-SP4 autoantibodies by ELISA. A validation cohort of 46 anti-TIF1γ-positive patient sera from the University of Pittsburgh was also screened for anti-Sp4 autoantibodies. RESULTS: Anti-Sp4 autoantibodies were present in 27 (10.5%) patients with DM and 1 (3.3%) patient with RA but not in other clinical groups. In patients with DM, 96.3% of anti-Sp4 autoantibodies were detected in those with anti-TIF1γ autoantibodies. Among 26 TIF1γ-positive patients with anti-Sp4 autoantibodies, none (0%) had cancer. In contrast, among 35 TIF1γ-positive patients without anti-Sp4 autoantibodies, 5 (14%, p=0.04) had cancer. In the validation cohort, among 15 TIF1γ-positive patients with anti-Sp4 autoantibodies, 2 (13.3%) had cancer. By comparison, among 31 TIF1γ-positive patients without anti-Sp4 autoantibodies, 21 (67.7%, p<0.001) had cancer. CONCLUSIONS: Anti-Sp4 autoantibodies appear to identify a subgroup of anti-TIF1γ-positive DM patients with lower cancer risk.

Phage display of environmental protein toxins and virulence factors reveals the prevalence, persistence, and genetics of antibody responses.

Microbial exposures are crucial environmental factors that impact healthspan by sculpting the immune system and microbiota. Antibody profiling via Phage ImmunoPrecipitation Sequencing (PhIP-Seq) provides a high-throughput, cost-effective approach for detecting exposure and response to microbial protein products. We designed and constructed a library of 95,601 56-amino acid peptide tiles spanning 14,430 proteins with "toxin" or "virulence factor" keyword annotations. We used PhIP-Seq to profile the antibodies of ∼1,000 individuals against this "ToxScan" library. In addition to enumerating immunodominant antibody epitopes, we studied the age-dependent stability of the ToxScan profile and used a genome-wide association study to find that the MHC-II locus modulates bacterial epitope selection. We detected previously described anti-flagellin antibody responses in a Crohn's disease cohort and identified an association between anti-flagellin antibodies and juvenile dermatomyositis. PhIP-Seq with the ToxScan library is thus an effective tool for studying the environmental determinants of health and disease at cohort scale.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Profiling serum antibodies with a pan allergen phage library identifies key wheat allergy epitopes.

Allergic reactions occur when IgE molecules become crosslinked by antigens such as food proteins. Here we create the 'AllerScan' programmable phage display system to characterize the binding specificities of anti-allergen IgG and IgE antibodies in serum against thousands of allergenic proteins from hundreds of organisms at peptide resolution. Using AllerScan, we identify robust anti-wheat IgE reactivities in wheat allergic individuals but not in wheat-sensitized individuals. Meanwhile, a key wheat epitope in alpha purothionin elicits dominant IgE responses among allergic patients, and frequent IgG responses among sensitized and non-allergic patients. A double-blind, placebo-controlled trial shows that alpha purothionin reactivity, among others, is strongly modulated by oral immunotherapy in tolerized individuals. AllerScan may thus serve as a high-throughput platform for unbiased analysis of anti-allergen antibody specificities.

Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity.

Understanding humoral responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics, therapeutics, and vaccines. Deep serological profiling of 232 coronavirus disease 2019 (COVID-19) patients and 190 pre-COVID-19 era controls using VirScan revealed more than 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies. Preexisting antibodies in controls recognized SARS-CoV-2 ORF1, whereas only COVID-19 patient antibodies primarily recognized spike protein and nucleoprotein. A machine learning model trained on VirScan data predicted SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-CoV-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-CoV-2 responses, weaker antibody responses to prior infections, and higher incidence of cytomegalovirus and herpes simplex virus 1, possibly influenced by demographic covariates. Among hospitalized patients, males produce stronger SARS-CoV-2 antibody responses than females.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen- (and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

LINE-1 ORF2p expression is nearly imperceptible in human cancers.

BACKGROUND: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. RESULTS: We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. CONCLUSIONS: Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

Comprehensive Profiling of HIV Antibody Evolution.

This study evaluates HIV antibody responses and their evolution during the course of HIV infection. A phage display system is used to characterize antibody binding to >3,300 HIV peptides in 57 adults with early- to late-stage infection. We find that the number of unique epitopes targeted ("antibody breadth") increases early in infection and then stabilizes or declines. A decline in antibody breadth 9 months to 2 years after infection is associated with subsequent antiretroviral treatment (ART) initiation, and a faster decline in antibody breadth is associated with a shorter time to ART initiation. We identify 266 peptides with increasing antibody reactivity over time and 43 peptides with decreasing reactivity over time. These data are used to design a prototype four-peptide "serosignature" to predict duration of HIV infection. We also demonstrate that epitope engineering can be used to optimize peptide binding properties for applications such as cross-sectional HIV incidence estimation.

Publisher Correction: PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes.

The version of this paper originally published contained typesetter-introduced errors in some of the code commands, consisting of conversion of a closing backslash (\) to a forward slash (/). These errors have been corrected in the HTML and PDF versions of the protocol.

PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes.

The binding specificities of an individual's antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing 'phage immunoprecipitation sequencing' (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost. The methodology uses oligonucleotide library synthesis (OLS) to encode proteomic-scale peptide libraries for display on bacteriophage. These libraries are then immunoprecipitated, using an individual's antibodies, for subsequent analysis by high-throughput DNA sequencing. We have used PhIP-Seq to identify novel self-antigens associated with autoimmune disease, to characterize the self-reactivity of broadly neutralizing HIV antibodies, and in a large international cross-sectional study of exposure to hundreds of human viruses. Compared with alternative array-based techniques, PhIP-Seq is far more scalable in terms of sample throughput and cost per analysis. Cloning and expression of recombinant proteins are not required (versus protein microarrays), and peptide lengths are limited only by DNA synthesis chemistry (up to 90-aa (amino acid) peptides versus the typical 8- to 12-aa length limit of synthetic peptide arrays). Compared with protein microarrays, however, PhIP-Seq libraries lack discontinuous epitopes and post-translational modifications. To increase the accessibility of PhIP-Seq, we provide detailed instructions for the design of phage-displayed peptidome libraries, their immunoprecipitation using serum antibodies, deep sequencing-based measurement of peptide abundances, and statistical determination of peptide enrichments that reflect antibody-peptide interactions. Once a library has been constructed, PhIP-Seq data can be obtained for analysis within a week.