- DEFEN-CE: Social Dialogue in Defence of Vulnerable Groups in Post-COVID-19 Labour Markets. Comparative report -
The COVID-19 pandemic has generated unprecedented health and far-reaching life consequences, triggering a global social and economic crisis through its protective measures aimed at safeguarding lives. This crisis compels social scientists and researchers to scrutinize the deficiencies in social and economic readiness and responses to the pandemic. The DEFEN-CE project, supported by the European Commission, delves into institutional strategies and power dynamics in social protection, policy formulation, and implementation. It sought to safeguard labour markets and workers by examining the governance of vulnerable groups in the (post) COVID-19 labor markets. Moreover, it aimed to generate research-based knowledge at EU and national levels, including candidate countries, on the role of social partners in creating and implementing protective policies vis-à-vis vulnerable groups. This report spotlights all key project findings both at the EU-level and the national level in 12 countries, embedding them to a conceptual understanding of vulnerability in general and labour-market related vulnerability in particular.
- DEFEN-CE: Social Dialogue in Defence of Vulnerable Groups in Post-COVID-19 LabourMarkets. EU-wide analysis of the Defence - database data -
The DEFEN-CE project’s Defence Database safeguards vulnerable groups in post-COVID-19 European labour markets. The research teams analysed data from EU-27 Member States, Turkey, and Serbia, including indicators covering policy, target groups, and social partners’ involvement. The analysis includes 853 policies.
- Spatial Morphoproteomic Features Predict Uniqueness of Immune Microarchitectures and Responses in Lymphoid Follicles -
Multiplex imaging technologies allow the characterization of single cells in their cellular environments. Understanding the organization of single cells within their microenvironment and quantifying disease-status related biomarkers is essential for multiplex datasets. Here we proposed SNOWFLAKE, a graph neural network framework pipeline for the prediction of disease-status from combined multiplex cell expression and morphology in human B-cell follicles. We applied SNOWFLAKE to a multiplex dataset related to COVID-19 infection in humans and showed better predictive power of the SNOWFLAKE pipeline compared to other machine learning and deep learning methods. Moreover, we combined morphological features inside graph edge features to utilize attribution methods for extracting disease-relevant motifs from single-cell spatial graphs. The underlying subgraphs were further analyzed and associated with disease status across the dataset. We showed that SNOWFLAKE successfully extracted significant low dimensional embedding from subgraphs with a clear separation between disease status and helped characterize unique cellular interactions in the subgraphs. SNOWFLAKE is a generalizable pipeline for the analysis of multiplex imaging data modality by extracting disease-relevant subgraphs guided by graph-level prediction.
- CGRP inhibits SARS-CoV-2 infection of bronchial epithelial cells and its pulmonary levels correlate with viral clearance in critical COVID-19 patients -
Upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), patients with critical coronavirus disease 2019 (COVID-19) present with life-threatening respiratory distress, pulmonary damage and cytokine storm. One unexplored hub in COVID-19 is the neuropeptide calcitonin gene-related peptide (CGRP), which is highly abundant in the airways and could converge in multiple aspects of COVID-19-related pulmonary pathophysiology. Whether CGRP affects SARS-CoV-2 infection directly remains elusive. We show that in critical COVID-19 patients, CGRP is increased in both plasma and lungs. Importantly, CGRP pulmonary levels are elevated in early SARS-CoV-2-positive patients, and restore to baseline upon subsequent viral clearance in SARS-CoV-2-negative patients. We further show that CGRP and its stable analogue SAX directly inhibit infection of bronchial Calu-3 epithelial cells with SARS-CoV-2 Omicron and Alpha variants in a dose-dependent manner. Both pre- and post-infection treatment with GRRP and/or SAX is enough to block SARS-CoV-2 productive infection of Calu3 cells. CGRP-mediated inhibition occurs via activation of the CGRP receptor and involves down-regulation of SARS-CoV-2 entry receptors at the surface of Calu-3 cells. Together, we propose that increased pulmonary CGRP mediates beneficial viral clearance in critical COVID-19 patients, by directly inhibiting SARS-CoV-2 infection. Hence, CGRP-based interventions could be harnessed for management of COVID-19.
- Rapid Emergence and Evolution of SARS-CoV-2 Variants in Advanced HIV Infection -
Previous studies have linked the evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic variants to persistent infections in people with immunocompromising conditions, but the evolutionary processes underlying these observations are incompletely understood. Here we used high-throughput, single-genome amplification and sequencing (HT-SGS) to obtain up to ~103 SARS-CoV-2 spike gene sequences in each of 184 respiratory samples from 22 people with HIV (PWH) and 25 people without HIV (PWOH). Twelve of 22 PWH had advanced HIV infection, defined by peripheral blood CD4 T cell counts (i.e., CD4 counts) <200 cells/L. In PWOH and PWH with CD4 counts [≥]200 cells/L, most single-genome spike sequences in each person matched one haplotype that predominated throughout the infection. By contrast, people with advanced HIV showed elevated intra-host spike diversity with a median of 46 haplotypes per person (IQR 14-114). Higher intra-host spike diversity immediately after COVID-19 symptom onset predicted longer SARS-CoV-2 RNA shedding among PWH, and intra-host spike diversity at this timepoint was significantly higher in people with advanced HIV than in PWOH. Composition of spike sequence populations in people with advanced HIV fluctuated rapidly over time, with founder sequences often replaced by groups of new haplotypes. These population-level changes were associated with a high total burden of intra-host mutations and positive selection at functionally important residues. In several cases, delayed emergence of detectable serum binding to spike was associated with positive selection for presumptive antibody-escape mutations. Taken together, our findings show remarkable intra-host genetic diversity of SARS-CoV-2 in advanced HIV infection and suggest that adaptive intra-host SARS-CoV-2 evolution in this setting may contribute to the emergence of new variants of concern (VOCs).
- Antigen-display exosomes provide adjuvant-free protection against SARS-CoV-2 disease at nanogram levels of spike protein -
As the only bionormal nanovesicle, exosomes have high potential as a nanovesicle for delivering vaccines and therapeutics. We show here that the loading of type-1 membrane proteins into the exosome membrane is induced by exosome membrane anchor domains, EMADs, that maximize protein delivery to the plasma membrane, minimize protein sorting to other compartments, and direct proteins into exosome membranes. Using SARS-CoV-2 spike as an example and EMAD13 as our most effective exosome membrane anchor, we show that cells expressing a spike-EMAD13 fusion protein produced exosomes that carry dense arrays of spike trimers on 50% of all exosomes. Moreover, we find that immunization with spike-EMAD13 exosomes induced strong neutralizing antibody responses and protected hamsters against SARS-CoV-2 disease at doses of just 0.5-5 ng of spike protein, without adjuvant, demonstrating that antigen-display exosomes are particularly immunogenic, with important implications for both structural and expression-dependent vaccines.
- Longitudinal transcriptional changes reveal genes from the natural killer cell-mediated cytotoxicity pathway as critical players underlying COVID-19 progression -
Patients present a wide range of clinical severities in response SARS-CoV-2 infection, but the underlying molecular and cellular reasons why clinical outcomes vary so greatly within the population remains unknown. Here, we report that negative clinical outcomes in severely ill patients were associated with divergent RNA transcriptome profiles in peripheral immune cells compared with mild cases during the first weeks after disease onset. Protein-protein interaction analysis indicated that early-responding cytotoxic NK cells were associated with an effective clearance of the virus and a less severe outcome. This innate immune response was associated with the activation of select cytokine-cytokine receptor pathways and robust Th1/Th2 cell differentiation profiles. In contrast, severely ill patients exhibited a dysregulation between innate and adaptive responses affiliated with divergent Th1/Th2 profiles and negative outcomes. This knowledge forms the basis of clinical triage that may be used to preemptively detect high-risk patients before life-threatening outcomes ensue.
- Revisit the Inhibitory Effects of Glucocorticoids on Immunocytes -
Glucocorticoids (GCs) are efficacious agents for reducing inflammation and suppressing immune responses, exerting various effects on immune cells through the intracellular glucocorticoid receptor (GR), and impacting both innate and adaptive immunity. In the context of COVID-19, glucocorticoids are often used to treat severe cases of patients by reducing inflammation, suppressing immune responses, and ameliorating the severity of COVID-19. However, the precise inhibitory effects on immune cells have yet to be comprehensively delineated. In this study, we extensively examined the inhibitory effects of treating Balb/c mice with dexamethasone (DEX) on lymphoid and myeloid cells. We observed that high doses of DEX treatment resulted in a reduction in the number of immunocytes and an attenuation of their activity. Particularly noteworthy, macrophages, DC cells, and monocytes were diminished by approximately 90% following high doses of DEX, while B cells experienced a reduction of about 70% and CD3 T cells were less affected. Furthermore, our findings demonstrated that DEX induces the inhibition of immune cells by engaging in high-affinity binding to GR. Consequently, we conclude that DEX treatments affect a broad range of immune cells, encompassing both lymphoid and myeloid cells, through depletion or the down-regulation of immune function, potentially acting via the GR signaling pathway. These findings may enhance the clinical applicability of DEX in achieving transient immune deficiency.
- Stably-Inverted Apical-Out Human Upper Airway Organoids for SARS-CoV-2 Infection and Therapeutic Testing -
Apical-out organoids produced through eversion triggered by extra-organoid extracellular matrix (ECM) removal or degradation are generally small, structurally variable, and limited for viral infection and therapeutics testing. This work describes ECM-encapsulating, stably-inverted apical-out human upper airway organoids (AORBs) that are large (~500 um diameter), consistently spherical, recapitulate in vivo-like cellular heterogeneity, and maintain their inverted morphology for over 60 days. Treatment of AORBs with IL-13 skews differentiation towards goblet cells and the apical-out geometry allows extra-organoid mucus collection. AORB maturation for 14 days induces strong co-expression of ACE2 and TMPRSS2 to allow high-yield infection with five SARS-CoV-2 variants. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds [remdesivir, bemnifosbuvir (AT-511), and nirmatrelvir] shows AORB antiviral assays to be comparable to gold-standard air-liquid interface cultures, but with higher throughput (~10-fold) and fewer cells (~100-fold). While this work focuses on SARS-CoV-2 applications, the consistent AORB shape and size, and one-organoid-per-well modularity broadly impacts in vitro human cell model standardization efforts in line with economic imperatives and recently updated FDA regulation on therapeutic testing.
- A speed limit on serial strain replacement from original antigenic sin -
Many pathogens evolve to escape immunity, yet it remains difficult to predict whether immune pressure will lead to diversification, serial replacement of one variant by another, or more complex patterns. Pathogen strain dynamics are mediated by cross-protective immunity, whereby exposure to one strain partially protects against infection by antigenically diverged strains. There is growing evidence that this protection is influenced by early exposures, a phenomenon referred to as original antigenic sin (OAS) or imprinting. In this paper, we derive new constraints on the emergence of the pattern of successive strain replacements demonstrated by influenza, SARS-CoV-2, seasonal coronaviruses, and other pathogens. We find that OAS implies that the limited diversity characteristic of successive strain replacement can only be maintained if R0 is less than a threshold set by the characteristic antigenic distances for cross-protection and for the creation of new immune memory. This bound implies a "speed limit" on the evolution of new strains and a minimum variance of the distribution of infecting strains in antigenic space at any time. To carry out this analysis, we develop a theoretical model of pathogen evolution in antigenic space that implements OAS by decoupling the antigenic distances required for protection from infection and strain-specific memory creation. Our results demonstrate that OAS can play an integral role in the emergence of strain structure from host immune dynamics, preventing highly transmissible pathogens from maintaining serial strain replacement without diversification.
- Lethal Infection of Human ACE2-Transgenic Mice Caused by SARS-CoV-2-related Pangolin Coronavirus GX_P2V(short_3UTR) -
SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) can cause 100% mortality in human ACE2-transgenic mice, potentially attributable to late-stage brain infection. This underscores a spillover risk of GX_P2V into humans and provides a unique model for understanding the pathogenic mechanisms of SARS-CoV-2-related viruses.
- A Murine Model of Post-acute Neurological Sequelae Following SARS-CoV-2 Variant Infection -
Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. Surviving mice showed no detectable viral RNA in the brain and minimal neuroinflammation post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and reduced levels of genes associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent T helper 1 prone cellular immune responses and high neutralizing antibodies against Delta and Omicron variants in the periphery for months post-acute infection. Overall, infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long COVID patients and may be useful for future assessment of the efficacy of vaccines and therapeutics against SARS-CoV-2 variants.
- Prototype mRNA vaccines imprint broadly neutralizing human serum antibodies after Omicron variant-matched boosting -
Immune imprinting is a phenomenon in which an individual's prior antigenic experiences influence responses to subsequent infection or vaccination. Here, using antibody depletion and multiplexed spike-binding assays, we characterized the type-specificity and cross-reactivity of serum antibody responses after mRNA vaccination in mice and human clinical trial participants. In mice, a single priming dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike minimally imprinted serum responses elicited by Omicron boosters, enabling a robust generation of type-specific antibodies. However, substantial imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron mRNA vaccine. In humans who received two BA.5 or XBB.1.5 Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with circulating Omicron variants as well as more distantly related sarbecoviruses. Because the serum neutralizing response against Omicron strains and other sarbecoviruses was completely abrogated after pre-clearing with the Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes shaped and shared by the antecedent mRNA-1273 primary series. Our depletion analysis also identified cross-reactive neutralizing antibodies that recognize distinct epitopes in the receptor binding domain (RBD) and S2 proteins with differential inhibitory effects on members of the sarbecovirus subgenus. Thus, although the serum antibody response to Omicron-based boosters in humans is dominantly imprinted by prior immunizations with prototype mRNA-1273 vaccines, this outcome can be beneficial as it drives expansion of multiple classes of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and extend activity to distantly related sarbecoviruses.
- Change in Anti-COVID-19 Behavior and Prejudice against Minorities during the COVID-19 Pandemic: Longitudinal Evidence from Five European Countries -
In the COVID-19 pandemic, it is vital to identify factors increasing behaviors that limit the transmission of COVID-19 (i.e., anti-COVID-19 behavior) and factors protecting against the negative consequences of the pandemic on societies (i.e., prejudice). A simultaneous investigation of a change in anti-COVID behavior and prejudice during the pandemic is essential because some factors (e.g., fear of COVID-19) could increase both outcomes, whilst other factors (e.g., norms in anti-COVID behavior or intergroup contact in prejudice) could bring desirable changes in one outcome without negatively affecting the other. In a three-wave longitudinal study (NT1 = 4275) in five European countries from April to October 2020, we employed a latent change score model to distinguish between intra- and inter-individual changes in anti-COVID-19 behavior and prejudice. On the intra-individual level, anti-COVID-19 behavior was increased by anti-COVID-19 norms; and prejudice against migrants from the Middle East was influenced by positive and negative direct and mass-media intergroup contact.
- Mutation of highly conserved residues in loop 2 of the coronavirus macrodomain demonstrates that enhanced ADP-ribose binding is detrimental to infection -
All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication and pathogenesis.