B and T Cell Responses to SARS-CoV-2 Vaccination in Kidney and Liver Transplant Recipients with and without Previous COVID-19.

(1) Background: Vulnerable populations including transplant recipients are jeopardised by COVID-19. Herein, we report on B and T cell responses among liver and kidney organ recipients at our centre. (2) Methods: 23 liver and 45 kidney (14 thereof combined kidney/pancreas) transplanted patients were vaccinated with two doses of BNT162b2 followed by a booster dose of mRNA-1273 in 28 non-responders 4 months thereafter. Anti-SARS-CoV-2-Ig was measured by specific ELISA and virus neutralisation assay; T cell responses were measured by a spike protein-specific IFN-γ release assay. (3) Results: Compared to controls, B and T cell responses were weak in transplant recipients, particularly in those without prior exposure to SARS-CoV-2. Within this group, only 15% after the first and 58.3% after the second vaccination achieved seroconversion. A total of 14 out of 28 vaccination non-responders achieved a seroconversion after a third dose. Vaccination side effects were more frequent in healthy controls. The use of mycophenolate was associated with reduced anti-SARS-CoV-2-Ig production. (4) Conclusions: Our data confirm that vaccination responses are insufficient after standard vaccination in liver and kidney transplant recipients and are affected to a variable degree by specific immunosuppressants, particularly mycophenolate. Monitoring vaccination success and re-vaccinating those who are unresponsive seems prudent to achieve sufficient titres. Overall, prospective large-scale, multinational, multicentre studies or high-quality meta-analyses will be needed to generate personalised vaccination strategies in order to achieve protective immunity in high-risk, hard-to-immunize populations.

B and T cell response to SARS-CoV-2 vaccination in health care professionals with and without previous COVID-19.

BACKGROUND: In recent months numerous health care professional acquired COVID-19 at the workplace resulting in significant shortages in medical and nursing staff. We investigated how prior COVID-19 affects SARS-CoV-2 vaccination and how such knowledge could facilitate frugal vaccination strategies. METHODS: In a cohort of 41 healthcare professionals with (n=14) and without (n=27) previous SARS-CoV-2 infection, we assessed the immune status before, during and after vaccination with BNT162b2. The humoral immune response was assessed by receptor binding domain ELISA and different SARS-CoV-2 neutralisation assays using wildtype and pseudo-typed viruses. T cell immunity against SARS-CoV-2 surface and nucleocapsid peptides were studied using interferon-γ release assays and intracellular flow cytometry. Vaccine-related side effects were captured. FINDINGS: Prior COVID-19 resulted in improved vaccine responses both in the B and T cell compartment. In vaccine recipients with prior COVID-19, the first vaccine dose induced high antibody concentrations comparable to seronegative vaccine recipients after two injections. This translated into more efficient neutralisation of virus particles, even more pronounced than expected from the RBD ELISA results. Furthermore, T cell responses were stronger in convalescents and particularly strong against the SARS-CoV-2 nucleocapsid protein. INTERPRETATION: Herein, we corroborate recent findings suggesting that in convalescents a single vaccine dose is sufficient to boost adequate in vitro neutralisation of SARS-CoV-2 and therefore may be sufficient to induce adequate protection against severe COVID-19. New spike mutated virus variants render the highly conserved nucleocapsid protein - eliciting strong SARS-CoV-2 specific T cell immunity - an interesting additional vaccine target. FUNDING: Christian Doppler Research Association, Johannes Kepler University Linz.

Prevalence of Atrial Fibrillation and Antithrombotic Therapy in Hemodialysis Patients: Cross-Sectional Results of the Vienna InVestigation of AtriaL Fibrillation and Thromboembolism in Patients on HemoDIalysis (VIVALDI).

BACKGROUND: Atrial fibrillation (AF) adds significant risk of stroke and thromboembolism in patients on hemodialysis (HD). The aim of this study was to investigate the prevalence of AF in a population-based cohort of HD patients and practice patterns of antithrombotic therapy for stroke prevention in AF. METHODS: The Vienna InVestigation of AtriaL fibrillation and thromboembolism in patients on hemodialysis (VIVALDI), an ongoing prospective observational cohort study, investigates the prevalence of AF and the risk of thromboembolic events in HD patients in Vienna, Austria. We analyzed cross-sectional data of 626 patients (63.4% men, median age 66 years, approx. 73% of HD patients in Vienna), who provided informed consent. A structured interview with each patient was performed, recent and archived ECGs were viewed and medical histories were verified with electronic records. RESULTS: The overall prevalence of AF was 26.5% (166 patients, 71.1% men, median age 72 years) of which 57.8% had paroxysmal AF, 3.0% persistent AF, 32.5% permanent AF, and 6.6% of patients had newly diagnosed AF. The median CHA2DS2-VASc Score was 4 [25th-75th percentile 3-5]. In multivariable analysis, AF was independently associated with age (odds ratio: 1.05 per year increase, 95% confidence interval: 1.03-1.07), male sex (1.7, 1.1-2.6), history of venous thromboembolism (2.0, 1.1-3.6), congestive heart failure (1.7, 1.1-2.5), history of or active cancer (1.5, 1.0-2.4) and time on HD (1.08 per year on HD, 1.03-1.13). Antithrombotic treatment was applied in 84.4% of AF patients (anticoagulant agents in 29.5%, antiplatelet agents in 33.7%, and both in 21.1%). In AF patients, vitamin-K-antagonists were used more often than low-molecular-weight heparins (30.1% and 19.9%). CONCLUSIONS: The prevalence of AF is high amongst HD patients and is associated with age, sex, and distinct comorbidities. Practice patterns of antithrombotic treatment indicate a lack of consensus for stroke prevention in HD patients with AF.

Transcriptional regulators form diverse groups with context-dependent regulatory functions.

One of the most important questions in biology is how transcription factors (TFs) and cofactors control enhancer function and thus gene expression. Enhancer activation usually requires combinations of several TFs, indicating that TFs function synergistically and combinatorially. However, while TF binding has been extensively studied, little is known about how combinations of TFs and cofactors control enhancer function once they are bound. It is typically unclear which TFs participate in combinatorial enhancer activation, whether different TFs form functionally distinct groups, or if certain TFs might substitute for each other in defined enhancer contexts. Here we assess the potential regulatory contributions of TFs and cofactors to combinatorial enhancer control with enhancer complementation assays. We recruited GAL4-DNA-binding-domain fusions of 812 Drosophila TFs and cofactors to 24 enhancer contexts and measured enhancer activities by 82,752 luciferase assays in S2 cells. Most factors were functional in at least one context, yet their contributions differed between contexts and varied from repression to activation (up to 289-fold) for individual factors. Based on functional similarities across contexts, we define 15 groups of TFs that differ in developmental functions and protein sequence features. Similar TFs can substitute for each other, enabling enhancer re-engineering by exchanging TF motifs, and TF-cofactor pairs cooperate during enhancer control and interact physically. Overall, we show that activators and repressors can have diverse regulatory functions that typically depend on the enhancer context. The systematic functional characterization of TFs and cofactors should further our understanding of combinatorial enhancer control and gene regulation.

Genome-scale functional characterization of Drosophila developmental enhancers in vivo.

Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.

Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features.

Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancers' cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.

Transcriptional enhancers: from properties to genome-wide predictions.

Cellular development, morphology and function are governed by precise patterns of gene expression. These are established by the coordinated action of genomic regulatory elements known as enhancers or cis-regulatory modules. More than 30 years after the initial discovery of enhancers, many of their properties have been elucidated; however, despite major efforts, we only have an incomplete picture of enhancers in animal genomes. In this Review, we discuss how properties of enhancer sequences and chromatin are used to predict enhancers in genome-wide studies. We also cover recently developed high-throughput methods that allow the direct testing and identification of enhancers on the basis of their activity. Finally, we discuss recent technological advances and current challenges in the field of regulatory genomics.

cis-regulatory requirements for tissue-specific programs of the circadian clock.

BACKGROUND: Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs CLOCK (CLK) and CYCLE (CYC) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how CLK and CYC determine tissue-specific clock programs has remained unclear. RESULTS: Here, we use a functional genomics approach to determine the cis-regulatory requirements for clock specificity. We first determine CLK and CYC genome-wide binding targets in heads and bodies by ChIP-seq and show that they have distinct DNA targets in the two tissue contexts. Computational dissection of CLK/CYC context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, we show that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by OPA and SERPENT (SRP). They act synergistically with CLK/CYC in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, we validate that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock. CONCLUSIONS: Our results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression.

HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature.

HOT (highly occupied target) regions bound by many transcription factors are considered to be one of the most intriguing findings of the recent modENCODE reports, yet their functions have remained unclear. We tested 108 Drosophila melanogaster HOT regions in transgenic embryos with site-specifically integrated transcriptional reporters. In contrast to prior expectations, we found 102 (94%) to be active enhancers during embryogenesis and to display diverse spatial and temporal patterns, reminiscent of expression patterns for important developmental genes. Remarkably, HOT regions strongly activate nearby genes and are required for endogenous gene expression, as we show using bacterial artificial chromosome (BAC) transgenesis. HOT enhancers have a distinct cis-regulatory signature with enriched sequence motifs for the global activators Vielfaltig, also known as Zelda, and Trithorax-like, also known as GAGA. This signature allows the prediction of HOT versus control regions from the DNA sequence alone.