SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs
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PROVIDER: PRJNA667921 | ENA |
REPOSITORIES: ENA
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Project description:T cell-mediated immunity seems to play a critical role against SARS-CoV-2 infection and establishing protective memory. Yet the repertoire of viral epitopes responsible for activation of T cell responses remains mostly unknown. Identification and characterization of viral peptides presented on class I human leukocyte antigen (HLA-I) will reveal the viral signatures as seen by cytotoxic T cells that can then be harnessed for the development of effective vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not currently captured by vaccine approaches. Whole proteome analysis revealed that expression of ubiquitination pathway proteins, and the proteasome maturation protein, POMP, were significantly altered in infected cells. Retrospective analysis of computational predictions highlighted shortcomings of in silico-only approaches in recovering the observed viral epitopes. Finally, given the experimental evidence for endogenous processing and presentation of the viral peptides we detected, we estimated that at least one HLA-A, -B, or -C allele is covered by at least one peptide for 99% of the population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.
2020-10-08 | GSE159191 | GEO
Project description:Weingarten-Gabbay S, Klaeger S, Sarkizova S, Pearlman LR, Chen D, Gallagher K, Bauer MR, Taylor HB, W. Augustine Dunn WA, Tarr C, Sidney J, Rachimi S, Conway HL, Katsis K, Yuntong Wang Y, Leistritz-Edwards D, Durkin MR, Tomkins-Tinch CH, Finkel Y, Nachshon A, Gentili M, Rivera KD, Carulli IP, Chea VA, Chandrashekar A, Bozkus CC, Carrington M, MGH COVID-19 Collection & Processing Team, Bhardwaj N, Barouch DH, Sette A, Maus MV, Rice CM, Clauser KR, Keskin DB, Pregibon DC, Hacohen N, Carr SA, Abelin JG, Saeed M, Sabeti PC. 2021
T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection; yet the repertoire of naturally processed and presented viral epitopes on HLA class I remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and COVID-19 patients that exceeded responses to canonical peptides including some of the strongest epitopes reported to date. Whole proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights as well as the discovery of out-of-frame ORF epitopes will facilitate selection of peptides for immune monitoring and vaccine development.
2021-04-17 | MSV000087225 | MassIVE
Project description:Efforts to precisely identify tumor human leukocyte antigen presented peptides (HLAp) capable of mediating T cell based tumor rejection still face important challenges. Recent reports suggest that non-canonical cancer HLAp could be immunogenic but their identification requires highly sensitive and accurate mass-spectrometry (MS)-based proteogenomics approaches. Here, we present a novel MS-based analytical pipeline that can precisely characterize the non-canonical HLAp repertoire, incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLAp. Albeit often at low levels and in distinct subpopulations of cells, numerous non-canonical HLAp are shared across tumors. This analytical platform holds great promise for the discovery of novel cancer antigens for cancer immunotherapy.
2020-03-27 | PXD013649 | Pride
Project description:Seeking to identify HLA class I peptides that originate from vaccinia virus proteins to understand the mechanism of immune protection. Note that vaccinia-infected B cells will still continue to present (primarily) a wide variety of peptides originating from endogenous proteins; this data set contains evidence for more than 5000 such peptides. The objective and challenge is to detect and identify the peptides that originate from the pathogen (vaccinia virus) in the presence (background) of this large number of endogensous 'self' peptides. Keywords: Peptide search results from multiple injections of multiple strong cation exchange fractions combined into one set of results.
2009-08-31 | GSE10549 | GEO
Project description:Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the presentation and immunogenicity of nonC antigens across different cancer types to better understand their contribution to cancer immunosurveillance and to address whether they could be exploited therapeutically. To this end, we employed a proteogenomics pipeline to identify nonC HLA-I ligands derived from off-frame translation of coding sequences and non-coding regions (UTR, ncRNA, intronic and intergenic) in patient-derived tumor cell lines (TCL) of different histological types (4 gynecological cancer, 3 melanoma and 2 head and neck cancer patients). First, peptides bound to HLA-I were isolated and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using state-of-the-art procedures. Amino acid (Aa) sequences were identified through the previously described pipeline Peptide-PRISM, with some modifications. Briefly, for each MS spectrum, the top 10 candidates were first identified by de novo sequencing and later mapped to a database including the 3-frame transcriptome and 6-frame genome. Additionally, whole-exome sequencing (WES) information of each TCL was included to interrogate the presentation of mutated peptides derived cancer-specific NSM. The false-discovery rate (FDR) was calculated independently for each category considering the search space and peptide length in a stratified mixture model as previously described. Next, in order to select nonC peptides preferentially presented by tumor cells, immunopeptidomics data from several healthy tissues and samples available from the HLA ligand atlas was used to filter out peptides presented in healthy tissues. To overcome a potential bias toward frequent alleles, the peptides were excluded at the ORF level rather than the Aa sequence. As a result, we found that from a total of 839 unique nonC peptides detected in our tumor samples, 38.5% were predicted to derive from ORFs also present in healthy tissue (nonC-HL). Hence, 61.6% (n=517) were considered preferentially presented on tumor HLA-I, referred to as non-canonical tumor ligands (nonC-TL). Out results showed that, although nonC-TL constitued the most abundant source of candidate tumor antigens, as compared to neoantigens, cancer-germline or melanoma-associated antigens, pre-existing antitumor T cells in cancer patients preferentially recognized neoantigens rather than nonC-TL. Nonetehless, nonC-TL elicited de novo T-cell responses via in vitro sensitization of donor lymphocytes. We identified TCRs specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of the C5orf22C. These immunogenic nonC-TL were expressed across tumor types but were barely or not detected in healthy cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they are attractive novel targets for widely applicable immunotherapies.
2023-02-03 | PXD036856 | Pride
Project description:Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides but lacking canonical coding sequences. Apparently unable to produce peptides, lncRNA function seems to only involve RNA sequence and structure. Here, we exhaustively detect in-vivo translation of small open reading frames (small ORFs) within lncRNAs using Ribosomal profiling during Drosophila melanogaster embryogenesis. We show that around 30% of lncRNAs contain small ORFs engaged by ribosomes, leading to regulated translation of 100 to 300 micropeptides. We identify lncRNA features that favour translation, such as cistronicity, Kozak sequences, and conservation. For this latter, we develop a bioinformatics pipeline to detect small ORF homologues, and we reveal evidence of natural selection favouring the conservation of micropeptide sequence and function across evolution. Our results expand the repertoire of lncRNA functions, and suggest that lncRNAs give rise to novel coding genes throughout evolution. Since most lncRNAs contain small ORFs with as yet unknown translation potential, we propose to rename them “long non-canonical RNAs”.
2022-11-02 | GSE204739 | GEO
Project description:Immunopeptides that are translated in cells and presented at the cell surface by major histocompatibility complex (MHC) mole-cules are important epitopes in basic Immunology and translational cancer immunotherapy. While most of the reported immuno-peptides are derived from protein coding sequence, the non-canonical peptides translated from “non-coding” regions are emerging and have attracted much attention in recent years. However, sensitive and accurate identification of such peptides remains a challenging task. Here we report an optimized approach integrating Ribo-seq and mass spectrometry to identify hundreds of non-canonical MHC-binding peptides. Three pipelines for analyzing Ribo-seq data were compared to generate small open reading frame (sORF) databases. Meanwhile, we have also combined bottom-up and de novo searching in proteomics data analysis and identified more immunopeptides. 7902 canonical and 308 non-canonical immunopeptides have been identified with selected ones vigorously validated. The present study provides a handy solution for identifying non-canonical MHC epitopes. The novel im-munopeptides resolving mechanisms of cancer antigen presentation, as well as applications in cancer immunotherapies.
2021-11-03 | PXD024415 | Pride
Project description:Non-canonical peptides (NCPs) have been shown to play critical roles in fundamental biological processes. Despite their importance, NCPs have been rarely identified and characterized in plants. Here we developed an integrated peptidogenomic pipeline using high-throughput mass spectra to probe customized six-frame translation database and for the first time applied this pipeline to large-scale identification of NCPs in both monocot and dicot plants. Altogether, 1,993 and 1,860 NCPs were unambiguously identified in maize and Arabidopsis, respectively. The NCPs showed distinct characteristics compared to canonical peptides and can be derived from introns, 3’UTRs, 5’UTRs, junctions and intergenic regions. These results revealed that non-canonical translation or cryptic translation events occur more universally than previously thought. In addition, maize NCPs were found enriched within the regions associated with phenotypic variation or regions under domestication selection, indicating their potential function in plant genetic regulations of complex traits and evolution. Summarily, this study provides the first unbiased and global view of plant NCPs. The identification of large-scale NCPs in both monocot and dicot plants reveals that a much larger portion of the plant genome can be translated biologically functional molecules, which has important implications in functional genomic studies. The present study also provides a useful resource for the characterization of more hidden NCPs in other plants.
2020-08-11 | PXD017081 | Pride
Project description:We describe the identification of novel non-canonical ORFs in murine macrophages
2018-12-14 | GSE120762 | GEO
Project description:Studies have revealed dozens of functional peptides in putative ‘noncoding’ regions and raised the question of how many proteins are encoded by noncanonical open reading frames (ORFs). We comprehensively annotated genome-wide translated ORFs across five eukaryotic by analyzing ribosome profiling reads. We revealed pervasive translation in annotated untranslated regions of mRNAs or off-frame regions (i.e., uORFs, dORFs, and iORFs). We developed a logistic regression model based on ORF features to calculate the peptide functional probability in humans. Systematic ectopic expression experiments showed that peptides encoded by ncORFs follow the basic principles governing conventional protein subcellular localization and carry out diverse biological functions.
2024-05-23 | PXD037658 | Pride