Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

Proteasomes play a central role in the major histocompatibility class I (MHCI) antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site). There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. RESULTS: We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC), optimal cleavage models for the proteasome (MCC = 0.43 +/- 0.07) and the immunoproteasome (MCC = 0.36 +/- 0.06) were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. CONCLUSIONS: We have developed models that are specific to predict cleavage by the proteasome and the immunoproteasome. These models ought to be instrumental to identify protective CD8 T cell epitopes and are readily available for free public use at http://imed.med.ucm.es/Tools/PCPS

Selection of conserved epitopes from hepatitis C virus for pan-populational stimulation of T-cell responses

The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server

Computational assembly of a human Cytomegalovirus vaccine upon experimental epitope legacy

Background: Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus affecting approximately 90% of the world population. HCMV causes disease in immunologically naive and immunosuppressed patients. The prevention, diagnosis and therapy of HCMV infection are thus crucial to public health. The availability of effective prophylactic and therapeutic treatments remain a significant challenge and no vaccine is currently available. Here, we sought to define an epitope-based vaccine against HCMV, eliciting B and T cell responses, from experimentally defined HCMV-specific epitopes. Results: We selected 398 and 790 experimentally validated HCMV-specific B and T cell epitopes, respectively, from available epitope resources and apply a knowledge-based approach in combination with immunoinformatic predictions to ensemble a universal vaccine against HCMV. The T cell component consists of 6 CD8 and 6 CD4 T cell epitopes that are conserved among HCMV strains. All CD8 T cell epitopes were reported to induce cytotoxic activity, are derived from early expressed genes and are predicted to provide population protection coverage over 97%. The CD4 T cell epitopes are derived from HCMV structural proteins and provide a population protection coverage over 92%. The B cell component consists of just 3 B cell epitopes from the ectodomain of glycoproteins L and H that are highly flexible and exposed to the solvent. Conclusions: We have defined a multiantigenic epitope vaccine ensemble against the HCMV that should elicit T and B cell responses in the entire population. Importantly, although we arrived to this epitope ensemble with the help of computational predictions, the actual epitopes are not predicted but are known to be immunogenic

EPIPOX:immunoinformatic characterization of the shared T-cell epitome between variola virus and related pathogenic orthopoxviruses

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes

Characterization of Conserved and Promiscuous Human Rhinovirus CD4 T Cell Epitopes

This research was supported by UCM research special funds to P.A.R. and by the CAM research agency through grant IND2020/BMD-17364 to P.A.R.Human rhinovirus (RV) is the most common cause of upper respiratory infections and exacerbations of asthma. In this work, we selected 14 peptides (6 from RV A and 8 from RV C) encompassing potential CD4 T cell epitopes. Peptides were selected for being highly conserved in RV A and C serotypes and predicted to bind to multiple human leukocyte antigen class II (HLA II) molecules. We found positive T cell recall responses by interferon gamma (IFNγ)-ELISPOT assays to eight peptides, validating seven of them (three from RV A and four from RV C) as CD4 T cell epitopes through intracellular cytokine staining assays. Additionally, we verified their promiscuous binding to multiple HLA II molecules by quantitative binding assays. According to their experimental HLA II binding profile, the combination of all these seven epitopes could be recognized by >95% of the world population. We actually determined IFNγ responses to a pool encompassing these CD4 T cell epitopes by intracellular cytokine staining, finding positive responses in 29 out of 30 donors. The CD4 T cell epitopes identified in this study could be key to monitor RV infections and to develop peptide-based vaccines against most RV A and C serotypes.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEComunidad de MadridUniversidad Complutense de Madridpu

Cellular integrin ¿5ß1 and exosomal adam17 mediate the binding and uptake of exosomes produced by colorectal carcinoma cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by sizeexclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin 5 1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosome

Combining different bacteria in vaccine formulations enhances the chance for antiviral cross-reactive immunity: a detailed in silico analysis for influenza A virus

Bacteria are well known to provide heterologous immunity against viral infections through various mechanisms including the induction of innate trained immunity and adaptive cross-reactive immunity. Cross-reactive immunity from bacteria to viruses is responsible for long-term protection and yet its role has been downplayed due the difficulty of determining antigen-specific responses. Here, we carried out a systematic evaluation of the potential cross-reactive immunity from selected bacteria known to induce heterologous immunity against various viruses causing recurrent respiratory infections. The bacteria selected in this work were Bacillus Calmette Guerin and those included in the poly-bacterial preparation MV130: Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Klebisella pneumoniae, Branhamella catarrhalis and Haemophilus influenzae. The virus included influenza A and B viruses, human rhinovirus A, B and C, respiratory syncytial virus A and B and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through BLAST searches, we first identified the shared peptidome space (identity ≥ 80%, in at least 8 residues) between bacteria and viruses, and subsequently predicted T and B cell epitopes within shared peptides. Interestingly, the potential epitope spaces shared between bacteria in MV130 and viruses are non-overlapping. Hence, combining diverse bacteria can enhance cross-reactive immunity. We next analyzed in detail the cross-reactive T and B cell epitopes between MV130 and influenza A virus. We found that MV130 contains numerous cross-reactive T cell epitopes with high population protection coverage and potentially neutralizing B cell epitopes recognizing hemagglutinin and matrix protein 2. These results contribute to explain the immune enhancing properties of MV130 observed in the clinic against respiratory viral infections

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes

Guia de lípids i risc cardiovascular

Colesterol; Risc coronari; HipercolesterolèmiaCholesterol; Coronary risk; HypercholesterolemiaColesterol; Riesgo coronario; HipercolesterolemiaL’objectiu general d’aquesta guia és disposar d’unes recomanacions basades en l’evidència científica sobre el maneig dels lípids segons el risc cardiovascular (RCV). Aquesta guia engloba tot el procés assistencial i inclou els objectius comuns per tal d’augmentar la qualitat assistencial i disminuir la variabilitat assistencial en l’abordatge dels lípids i l’RCV