Characterization of SARS-CoV-2 public CD4+ αβ T cell clonotypes through reverse epitope discovery

The amount of scientific data and level of public sharing produced as a consequence of the COVID-19 pandemic, as well as the speed at which these data were produced, far exceeds any previous effort against a specific disease condition. This unprecedented situation allows for development and application of new research approaches. One of the major technical hurdles in immunology is the characterization of HLA-antigen-T cell receptor (TCR) specificities. Most approaches aim to identify reactive T cells starting from known antigens using functional assays. However, the need for a reverse approach identifying the antigen specificity of orphan TCRs is increasing. Utilizing large public single-cell gene expression and TCR datasets, we identified highly public CD4+ T cell responses to SARS-CoV-2, covering >75% of the analysed population. We performed an integrative meta-analysis to deeply characterize these clonotypes by TCR sequence, gene expression, HLA-restriction, and antigen-specificity, identifying strong and public CD4+ immunodominant responses with confirmed specificity. CD4+ COVID-enriched clonotypes show T follicular helper functional features, while clonotypes depleted in SARS-CoV-2 individuals preferentially had a central memory phenotype. In total we identify more than 1200 highly public CD4+ T cell clonotypes reactive to SARS-CoV-2. TCR similarity analysis showed six prominent TCR clusters, for which we predicted both HLA-restriction and cognate SARS-CoV-2 immunodominant epitopes. To validate our predictions we used an independent cohort of TCR repertoires before and after vaccination with ChAdOx1, a replication-deficient simian adenovirus-vectored vaccine, encoding the SARS-CoV-2 spike protein. We find statistically significant enrichment of the predicted spike-reactive TCRs after vaccination with ChAdOx1, while the frequency of TCRs specific to other SARS-CoV-2 proteins remains stable. Thus, the CD4-associated TCR repertoire differentiates vaccination from natural infection. In conclusion, our study presents a novel reverse epitope discovery approach that can be used to infer HLA- and antigen-specificity of orphan TCRs in any context, such as viral infections, antitumor immune responses, or autoimmune disease. HighlightsO_LIIdentification of highly public CD4+ T cell responses to SARS-CoV-2 C_LIO_LISystematic prediction of exact immunogenic HLA class II epitopes for CD4+ T cell response C_LIO_LIMethodological framework for reverse epitope discovery, which can be applied to other disease contexts and may provide essential insights for future studies and clinical applications C_LI O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

New susceptibility loci for severe COVID-19 by detailed GWAS analysis in European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic [~]0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.

Age-dependent impact of the major common genetic risk factor for COVID-19 on severity and mortality

BackgroundThere is considerable variability in COVID-19 outcomes amongst younger adults--and some of this variation may be due to genetic predisposition. We characterized the clinical implications of the major genetic risk factor for COVID-19 severity, and its age-dependent effect, using individual-level data in a large international multi-centre consortium. MethodThe major common COVID-19 genetic risk factor is a chromosome 3 locus, tagged by the marker rs10490770. We combined individual level data for 13,424 COVID-19 positive patients (N=6,689 hospitalized) from 17 cohorts in nine countries to assess the association of this genetic marker with mortality, COVID-19-related complications and laboratory values. We next examined if the magnitude of these associations varied by age and were independent from known clinical COVID-19 risk factors. FindingsWe found that rs10490770 risk allele carriers experienced an increased risk of all-cause mortality (hazard ratio [HR] 1{middle dot}4, 95% confidence interval [CI] 1{middle dot}2-1{middle dot}6) and COVID-19 related mortality (HR 1{middle dot}5, 95%CI 1{middle dot}3-1{middle dot}8). Risk allele carriers had increased odds of several COVID-19 complications: severe respiratory failure (odds ratio [OR] 2{middle dot}0, 95%CI 1{middle dot}6-2{middle dot}6), venous thromboembolism (OR 1{middle dot}7, 95%CI 1{middle dot}2-2{middle dot}4), and hepatic injury (OR 1{middle dot}6, 95%CI 1{middle dot}2-2{middle dot}0). Risk allele carriers [≤] 60 years had higher odds of death or severe respiratory failure (OR 2{middle dot}6, 95%CI 1{middle dot}8-3{middle dot}9) compared to those > 60 years OR 1{middle dot}5 (95%CI 1{middle dot}3-1{middle dot}9, interaction p-value=0{middle dot}04). Amongst individuals [≤] 60 years who died or experienced severe respiratory COVID-19 outcome, we found that 31{middle dot}8% (95%CI 27{middle dot}6-36{middle dot}2) were risk variant carriers, compared to 13{middle dot}9% (95%CI 12{middle dot}6-15{middle dot}2%) of those not experiencing these outcomes. Prediction of death or severe respiratory failure among those [≤] 60 years improved when including the risk allele (AUC 0{middle dot}82 vs 0{middle dot}84, p=0{middle dot}016) and the prediction ability of rs10490770 risk allele was similar to, or better than, most established clinical risk factors. InterpretationThe major common COVID-19 risk locus on chromosome 3 is associated with increased risks of morbidity and mortality--and these are more pronounced amongst individuals [≤] 60 years. The effect on COVID-19 severity was similar to, or larger than most established risk factors, suggesting potential implications for clinical risk management. FundingFunding was obtained by each of the participating cohorts individually.

Pre-existing T cell memory as a risk factor for severe 1 COVID-19 in the elderly

Coronavirus disease 2019 (COVID-19) displays high clinical variability but the parameters that determine disease severity are still unclear. Pre-existing T cell memory has been hypothesized as a protective mechanism but conclusive evidence is lacking. Here we demonstrate that all unexposed individuals harbor SARS-CoV-2-specific memory T cells with marginal cross-reactivity to common cold corona and other unrelated viruses. They display low functional avidity and broad protein target specificities and their frequencies correlate with the overall size of the CD4+ memory compartment reflecting the "immunological age" of an individual. COVID-19 patients have strongly increased SARS-CoV-2-specific inflammatory T cell responses that are correlated with severity. Strikingly however, patients with severe COVID-19 displayed lower TCR functional avidity and less clonal expansion. Our data suggest that a low avidity pre-existing T cell memory negatively impacts on the T cell response quality against neoantigens such as SARS-CoV-2, which may predispose to develop inappropriate immune reactions especially in the elderly. We propose the immunological age as an independent risk factor to develop severe COVID-19. Key points- Pre-existing SARS-CoV-2-reactive memory T cells are present in all humans, but have low functional avidity and broad target specificities - Pre-existing memory T cells show only marginal cross-reactivity to common cold corona viruses - Frequencies of pre-existing memory T cells increase with the size of the CD4+ memory compartment reflecting the "immunological age" of the individual - Low-avidity and polyclonal, but strongly enhanced SARS-CoV-2 specific T cell responses develop in severe COVID-19, suggesting their origin from pre-existing memory - The immunological age may represent a risk factor to develop severe COVID-19

Longitudinal multi-omics analysis identifies responses of megakaryocytes, erythroid cells and plasmablasts as hallmarks of severe COVID-19 trajectories

The pandemic spread of the potentially life-threatening disease COVID-19 requires a thorough understanding of the longitudinal dynamics of host responses. Temporal resolution of cellular features associated with a severe disease trajectory will be a pre-requisite for finding disease outcome predictors. Here, we performed a longitudinal multi-omics study using a two-centre German cohort of 13 patients (from Cologne and Kiel, cohort 1). We analysed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. The results from single-cell and bulk transcriptome analyses were validated in two independent cohorts of COVID-19 patients from Bonn (18 patients, cohort 2) and Nijmegen (40 patients, cohort 3), respectively. We observed an increase of proliferating, activated plasmablasts in severe COVID-19, and show a distinct expression pattern related to a hyperactive cellular metabolism of these cells. We further identified a notable expansion of type I IFN-activated circulating megakaryocytes and their progenitors, indicative of emergency megakaryopoiesis, which was confirmed in cohort 2. These changes were accompanied by increased erythropoiesis in the critical phase of the disease with features of hypoxic signalling. Finally, projecting megakaryocyte- and erythroid cell-derived co-expression modules to longitudinal blood transcriptome samples from cohort 3 confirmed an association of early temporal changes of these features with fatal COVID-19 disease outcome. In sum, our longitudinal multi-omics study demonstrates distinct cellular and gene expression dynamics upon SARS-CoV-2 infection, which point to metabolic shifts of circulating immune cells, and reveals changes in megakaryocytes and increased erythropoiesis as important outcome indicators in severe COVID-19 patients.

The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV-2 respiratory failure in an Italian-Spanish genome-wide association analysis

BackgroundRespiratory failure is a key feature of severe Covid-19 and a critical driver of mortality, but for reasons poorly defined affects less than 10% of SARS-CoV-2 infected patients. MethodsWe included 1,980 patients with Covid-19 respiratory failure at seven centers in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe (Milan, Monza, Madrid, San Sebastian and Barcelona) for a genome-wide association analysis. After quality control and exclusion of population outliers, 835 patients and 1,255 population-derived controls from Italy, and 775 patients and 950 controls from Spain were included in the final analysis. In total we analyzed 8,582,968 single-nucleotide polymorphisms (SNPs) and conducted a meta-analysis of both case-control panels. ResultsWe detected cross-replicating associations with rs11385942 at chromosome 3p21.31 and rs657152 at 9q34, which were genome-wide significant (P<5x10-8) in the meta-analysis of both study panels, odds ratio [OR], 1.77; 95% confidence interval [CI], 1.48 to 2.11; P=1.14x10-10 and OR 1.32 (95% CI, 1.20 to 1.47; P=4.95x10-8), respectively. Among six genes at 3p21.31, SLC6A20 encodes a known interaction partner with angiotensin converting enzyme 2 (ACE2). The association signal at 9q34 was located at the ABO blood group locus and a blood-group-specific analysis showed higher risk for A-positive individuals (OR=1.45, 95% CI, 1.20 to 1.75, P=1.48x10-4) and a protective effect for blood group O (OR=0.65, 95% CI, 0.53 to 0.79, P=1.06x10-5). ConclusionsWe herein report the first robust genetic susceptibility loci for the development of respiratory failure in Covid-19. Identified variants may help guide targeted exploration of severe Covid-19 pathophysiology.

Longitudinal high-throughput TCR repertoire profiling reveals the dynamics of T cell memory formation after mild COVID-19 infection

COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T cell response nor the diversity of resulting immune memory are well understood. In this study we use longitudinal high-throughput T cell receptor (TCR) sequencing to track changes in the T cell repertoire following two mild cases of COVID-19. In both donors we identified CD4+ and CD8+ T cell clones with transient clonal expansion after infection. The antigen specificity of CD8+ TCR sequences to SARS-CoV-2 epitopes was confirmed by both MHC tetramer binding and presence in large database of SARS-CoV-2 epitope-specific TCRs. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T cell clones were detected in the memory fraction at the pre-infection timepoint, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates / 基因组蛋白质组与生物信息学报·英文版

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.

Whither systems medicine?

New technologies to generate, store and retrieve medical and research data are inducing a rapid change in clinical and translational research and health care. Systems medicine is the interdisciplinary approach wherein physicians and clinical investigators team up with experts from biology, biostatistics, informatics, mathematics and computational modeling to develop methods to use new and stored data to the benefit of the patient. We here provide a critical assessment of the opportunities and challenges arising out of systems approaches in medicine and from this provide a definition of what systems medicine entails. Based on our analysis of current developments in medicine and healthcare and associated research needs, we emphasize the role of systems medicine as a multilevel and multidisciplinary methodological framework for informed data acquisition and interdisciplinary data analysis to extract previously inaccessible knowledge for the benefit of patients.

Stability, prognostic factors and survival of spinal bone metastases in malignant melanoma patients after palliative radiotherapy.

PURPOSE: This retrospective analysis aimed to evaluate the stability of spinal metastases in malignant melanoma patients following radiotherapy (RT), and to assess prognostic factors for survival. METHODS: Forty-one patients with malignant melanoma and osteolytic spinal bone metastases were irradiated at the university clinics of Heidelberg and Mainz between July 2003 and October 2013. Three and six months after palliative RT, only 20 and 15 patients, respectively, were still alive and were therefore assessed for spinal stability using the Taneichi score based on CT imaging. Additionally, overall survival (OS) and bone survival (BS) rates as well as prognostic factors for BS were evaluated for all study patients. RESULTS: Before RT, 19 patients (46.3%) were rated unstable. In the surviving patients, none of the unstable metastases were classified as stable 6 months after RT. Five-year OS was 23.3% and median BS was 4 months (range 0.5-29.8). Accordingly, only 36.6% of the patients were still alive 6 months after RT. Karnofsky performance score (KPS) <70%, visceral metastases and more than one bone metastasis were significantly predictive of poor BS. CONCLUSIONS: Our study population was characterized by poor BS and a lack of benefit with regard to stabilization of initially unstable spinal bone metastases 3 and 6 months after RT. This applies in particular to patients with a KPS <70%, visceral metastases and multiple bone metastases. Given the limited life expectancy, short fractionated treatment schedules of RT may be preferred in this population.

GLUT-1 expression is largely unrelated to both hypoxia and the Warburg phenotype in squamous cell carcinomas of the vulva.

BACKGROUND: Strongly increased uptake of glucose is a hallmark of solid malignant tumors. This phenotype can be triggered by hypoxia-induced gene expression changes or can occur independently of hypoxia as a consequence of malignant transformation itself, and is often referred to as the Warburg effect. The glycolytic phenotype has been associated with malignant progression and resistance to radio- and chemotherapy. METHODS: We have chosen squamous cell carcinomas of the vulva (SCC-V) as a representative solid tumor entity to study the central players of this pathway, namely glucose transporter (GLUT)-1, carbonic anhydrase (CA) IX, hexokinase (HK)-2 and pyruvate kinase (PK)-M2, and have investigated their relationships to tumor microvessels (CD34, αSMA) and proliferation (Ki67). Expression of these proteins was analyzed in 38 SCC-Vs, 5 vulvar dysplasias and 10 non-neoplastic squamous epithelia of the vulva using multiparametric immunohistochemistry in registered serial sections (MIRSS). RESULTS: Expression of GLUT-1 in invasive carcinomas was predominantly located in the outer layers of the tumor cell aggregates close to the vascularized tumor stroma, and only to a lesser extent colocalized with CA IX, which was repeatedly found at larger diffusion distances away from microvessels. CA IX expression was lower in invasive carcinomas compared to dysplasias and non-neoplastic tissue and higher in recurrent vs. primary tumors. Ki67-positive proliferating cells were partially colocalized with GLUT-1. However, HK-2 and PK-2--proteins centrally involved in the Warburg phenotype--did not show such a correlation. CONCLUSIONS: Consistent with prior studies, the pattern of GLUT-1 clearly indicated that a large part of its expression is presumably unrelated to hypoxia. However, there was also no association with HK-2 and PK-M2, suggesting that the functional background of this expression is also independent of aerobic glycolysis. CA IX may be worth consideration as a marker of biological hypoxia, as should its pathophysiological consequences in SCC-V.