We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open ...reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6
+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.
Pronounced immune escape by the SARS-CoV-2 Omicron variant has resulted in many individuals possessing hybrid immunity, generated through a combination of vaccination and infection. Concerns have ...been raised that omicron breakthrough infections in triple-vaccinated individuals result in poor induction of omicron-specific immunity, and that prior SARS-CoV-2 infection is associated with immune dampening. Taking a broad and comprehensive approach, we characterize mucosal and blood immunity to spike and non-spike antigens following BA.1/BA.2 infections in triple mRNA-vaccinated individuals, with and without prior SARS-CoV-2 infection. We find that most individuals increase BA.1/BA.2/BA.5-specific neutralizing antibodies following infection, but confirm that the magnitude of increase and post-omicron titres are higher in the infection-naive. In contrast, significant increases in nasal responses, including neutralizing activity against BA.5 spike, are seen regardless of infection history. Spike-specific T cells increase only in infection-naive vaccinees; however, post-omicron T cell responses are significantly higher in the previously-infected, who display a maximally induced response with a highly cytotoxic CD8+ phenotype following their 3
mRNA vaccine dose. Responses to non-spike antigens increase significantly regardless of prior infection status. These findings suggest that hybrid immunity induced by omicron breakthrough infections is characterized by significant immune enhancement that can help protect against future omicron variants.
Abstract
Hospital outbreaks of COVID19 result in considerable mortality and disruption to healthcare services and yet little is known about transmission within this setting. We characterise within ...hospital transmission by combining viral genomic and epidemiological data using Bayesian modelling amongst 2181 patients and healthcare workers from a large UK NHS Trust. Transmission events were compared between Wave 1 (1st March to 25th July 2020) and Wave 2 (30th November 2020 to 24th January 2021). We show that staff-to-staff transmissions reduced from 31.6% to 12.9% of all infections. Patient-to-patient transmissions increased from 27.1% to 52.1%. 40%-50% of hospital-onset patient cases resulted in onward transmission compared to 4% of community-acquired cases. Control measures introduced during the pandemic likely reduced transmissions between healthcare workers but were insufficient to prevent increasing numbers of patient-to-patient transmissions. As hospital-acquired cases drive most onward transmission, earlier identification of nosocomial cases will be required to break hospital transmission chains.
Abstract
Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug ...resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.
Inflammatory activation of monocytes is an essential part of both innate immune responses and the pathogenesis of conditions such as atherosclerosis. However, the mechanisms which modulate the ...response of monocytes to inflammatory stimuli are still poorly understood. Here, we report that tribbles-2 (trb-2) is a novel regulator of inflammatory activation of monocytes. Down-regulation of trb-2 levels potentiates LPS-induced IL-8 production via enhanced activation of the extracellular signal-regulated kinase and jun kinase mitogen-activated protein kinase (MAPK) pathways. In keeping with this, the endogenous level of trb-2 expression in human primary monocytes is inversely correlated to the cell’s ability to produce IL-8. We show that trb-2 is a binding partner and a negative regulator of selected MAPKs. The potential in vivo relevance of these findings is highlighted by the observation that modified low-density lipoprotein profoundly down-regulates trb-2 expression, which may, in turn, significantly contribute to the inflammatory processes in the development of vascular disease. Taken together, our results define trb-2 as a potent novel regulator of monocyte biology, controlling the activation of these cells.
The development of the SARS-CoV-2 pandemic has prompted an extensive worldwide sequencing effort to characterise the geographical spread and molecular evolution of the virus. A point mutation in the ...spike protein, D614G, emerged as the virus spread from Asia into Europe and the USA, and has rapidly become the dominant form worldwide. Here we review how the D614G variant was identified and discuss recent evidence about the effect of the mutation on the characteristics of the virus, clinical outcome of infection and host immune response.
The Grb2 associated binder (Gab) adaptor/scaffolding protein family comprises conserved proteins: mammalian Gab1, Gab2 and Gab3, Drosophila Dos and Caenorhabditis elegans Soc1. Gab adaptors are ...involved in multiple signaling pathways mediated by receptor- and non-receptor protein tyrosine kinases (PTKs), and become phosphorylated upon stimulation by growth factors-, cytokines-, Ig Fc- and antigen receptors. Through its phosphorylated tyrosine containing motifs, proline-rich sequences and pleckstrin homologue (PH) domain Gab adaptors may generate an interacting platform for proteins with SH2 and SH3 domains and may transfer these molecules to the plasma membrane, thereby contributing to their activation. This review will concentrate on the function of mammalian Gab proteins in the signal transduction triggered by immune receptors.
Fcγ receptors (FcγRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcγRs may facilitate antigen presentation and ...dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcγRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcγRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcγRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin–FITC, and tested its effect on the FITC–hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11bhigh and CD11chigh cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC–dextran) or TD (FITC–keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcγRIII might be a key mediator of this mechanism.
Extension of the interval between vaccine doses for the BNT162b2 mRNA vaccine was introduced in the United Kingdom to accelerate population coverage with a single dose. At this time, trial data were ...lacking, and we addressed this in a study of United Kingdom healthcare workers. The first vaccine dose induced protection from infection from the circulating alpha (B.1.1.7) variant over several weeks. In a substudy of 589 individuals, we show that this single dose induces severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody (NAb) responses and a sustained B and T cell response to the spike protein. NAb levels were higher after the extended dosing interval (6–14 weeks) compared with the conventional 3- to 4-week regimen, accompanied by enrichment of CD4+ T cells expressing interleukin-2 (IL-2). Prior SARS-CoV-2 infection amplified and accelerated the response. These data on dynamic cellular and humoral responses indicate that extension of the dosing interval is an effective immunogenic protocol.
Display omitted
•BNT162b2 vaccine with an extended interval between doses is highly protective•Antibody levels were higher after the extended regimen compared with the short regimen•The extended regimen enriches for virus-specific CD4+ T cells expressing IL-2•Antibody levels wane after each dose, but B and T cell pools are maintained
After giving a primary dose, delaying administration of a second dose of BNT162b2 COVID-19 vaccine up to 6–14 weeks continues to provide strong protection and contributes to favorable antibody, B cell, and T cell responses.
PDF
