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1.
PLoS Pathog ; 18(4): e1010464, 2022 Apr 14.
Article in English | MEDLINE | ID: covidwho-1789197

ABSTRACT

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

2.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-296014

ABSTRACT

ABSTRACT In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 ‘A’ strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine ( NCT04591717 ;https://clinicaltrials.gov/ct2/show/NCT04591717 ), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-292888

ABSTRACT

Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3′ (10X 3′) and Chromium Next GEM Single Cell V(D)J (10X 5′) sequencing, we find that 10X 5′ with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

4.
Non-conventional in English | [Unspecified Source], Grey literature | ID: grc-750490

ABSTRACT

Initially, the global outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spared children from severe disease. However, after the initial wave of infections, clusters of a novel hyperinflammatory disease have been reported in regions with ongoing SARS-CoV-2 epidemics. While the characteristic clinical features are becoming clear, the pathophysiology remains unknown. Herein, we report on the immune profiles of eight Multisystem Inflammatory Syndrome in Children (MIS-C) cases. We document that all MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with normal isotype-switching and neutralization capability. We further profiled the secreted immune response by high-dimensional cytokine assays, which identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1) and mucosal immune dysregulation (IL-17A, CCL20, CCL28). Mass cytometry immunophenotyping of peripheral blood revealed reductions of mDC1 and non-classical monocytes, as well as both NK- and T- lymphocytes, suggesting extravasation to affected tissues. Markers of activated myeloid function were also evident, including upregulation of ICAM1 and FcR1 in neutrophil and non-classical monocytes, well-documented markers in autoinflammation and autoimmunity that indicate enhanced antigen presentation and Fc-mediated responses. Finally, to assess the role for autoimmunity secondary to infection, we profiled the auto-antigen reactivity of MIS-C plasma, which revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal and immune-cell antigens. All patients were treated with anti- IL6R antibody or IVIG, which led to rapid disease resolution tracking with normalization of inflammatory markers.

5.
J Virol ; 95(23): e0125721, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1410202

ABSTRACT

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.


Subject(s)
COVID-19/metabolism , Cytokines/metabolism , Interferon Type I/metabolism , SARS-CoV-2 , Transcription Factor RelA/metabolism , Transcriptome , Virus Replication , A549 Cells , Animals , COVID-19/virology , Chlorocebus aethiops , Epigenomics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Host Microbial Interactions , Humans , Signal Transduction , Single-Cell Analysis , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/genetics , Transcription Factors/metabolism , Vero Cells
6.
Sci Rep ; 11(1): 12740, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1275953

ABSTRACT

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.


Subject(s)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , COVID-19/metabolism , Drug Discovery/methods , Molecular Dynamics Simulation , SARS-CoV-2/metabolism , Signal Transduction/drug effects , Amino Acid Sequence , COVID-19/virology , Humans , Mutation , Protein Binding/drug effects , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
7.
J Clin Immunol ; 41(7): 1457-1462, 2021 10.
Article in English | MEDLINE | ID: covidwho-1252168

ABSTRACT

While adults with Down syndrome (DS) are at increased risk of severe COVID-19 pneumonia, little is known about COVID-19 in children with DS. In children without DS, SARS-CoV-2 can rarely cause severe COVID-19 pneumonia, or an even rarer and more typically pediatric condition, multisystem inflammatory syndrome in children (MIS-C). Although the underlying mechanisms are still unknown, MIS-C is thought to be primarily immune-mediated. Here, we describe an atypical, severe form of MIS-C in two infant girls with DS who were hospitalized for over 4 months. Immunological evaluation revealed pronounced neutrophilia, B cell depletion, increased circulating IL-6 and IL-8, and elevated markers of immune activation ICAM1 and FcÉ£RI. Importantly, uninfected children with DS presented with similar but less stark immune features at steady state, possibly explaining risk of further uncontrolled inflammation following SARS-CoV-2 infection. Overall, a severe, atypical form of MIS-C may occur in children with DS.


Subject(s)
COVID-19/diagnosis , Down Syndrome/diagnosis , SARS-CoV-2/physiology , Systemic Inflammatory Response Syndrome/diagnosis , COVID-19/complications , Down Syndrome/complications , Fatal Outcome , Female , Hospitalization , Humans , Infant , Syndrome
8.
Proc Natl Acad Sci U S A ; 117(45): 28344-28354, 2020 11 10.
Article in English | MEDLINE | ID: covidwho-887237

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.


Subject(s)
COVID-19/metabolism , Interferons/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Binding Sites , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/metabolism , Protein Binding , Signal Transduction , Vero Cells
9.
Cell ; 183(4): 982-995.e14, 2020 11 12.
Article in English | MEDLINE | ID: covidwho-756809

ABSTRACT

Initially, children were thought to be spared from disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a month into the epidemic, a novel multisystem inflammatory syndrome in children (MIS-C) emerged. Herein, we report on the immune profiles of nine MIS-C cases. All MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with intact neutralization capability. Cytokine profiling identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1), and mucosal immune dysregulation (IL-17A, CCL20, and CCL28). Immunophenotyping of peripheral blood revealed reductions of non-classical monocytes, and subsets of NK and T lymphocytes, suggesting extravasation to affected tissues. Finally, profiling the autoantigen reactivity of MIS-C plasma revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal, and immune-cell antigens. All patients were treated with anti-IL-6R antibody and/or IVIG, which led to rapid disease resolution.


Subject(s)
Inflammation/pathology , Systemic Inflammatory Response Syndrome/pathology , Adolescent , Antibodies, Viral/blood , Autoantibodies/blood , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , COVID-19 , Chemokine CCL3/metabolism , Child , Child, Preschool , Coronavirus Infections/complications , Coronavirus Infections/pathology , Coronavirus Infections/virology , Female , Humans , Immunity, Humoral , Infant , Infant, Newborn , Inflammation/metabolism , Interleukin-17/metabolism , Interleukin-18/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Male , Pandemics , Pneumonia, Viral/complications , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , SARS-CoV-2 , Systemic Inflammatory Response Syndrome/immunology , Systemic Inflammatory Response Syndrome/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Young Adult
10.
medRxiv ; 2020 Jul 06.
Article in English | MEDLINE | ID: covidwho-663795

ABSTRACT

Initially, the global outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spared children from severe disease. However, after the initial wave of infections, clusters of a novel hyperinflammatory disease have been reported in regions with ongoing SARS-CoV-2 epidemics. While the characteristic clinical features are becoming clear, the pathophysiology remains unknown. Herein, we report on the immune profiles of eight Multisystem Inflammatory Syndrome in Children (MIS-C) cases. We document that all MIS-C patients had evidence of prior SARS-CoV-2 exposure, mounting an antibody response with normal isotype-switching and neutralization capability. We further profiled the secreted immune response by high-dimensional cytokine assays, which identified elevated signatures of inflammation (IL-18 and IL-6), lymphocytic and myeloid chemotaxis and activation (CCL3, CCL4, and CDCP1) and mucosal immune dysregulation (IL-17A, CCL20, CCL28). Mass cytometry immunophenotyping of peripheral blood revealed reductions of mDC1 and non-classical monocytes, as well as both NK- and T- lymphocytes, suggesting extravasation to affected tissues. Markers of activated myeloid function were also evident, including upregulation of ICAM1 and FcR1 in neutrophil and non-classical monocytes, well-documented markers in autoinflammation and autoimmunity that indicate enhanced antigen presentation and Fc-mediated responses. Finally, to assess the role for autoimmunity secondary to infection, we profiled the auto-antigen reactivity of MIS-C plasma, which revealed both known disease-associated autoantibodies (anti-La) and novel candidates that recognize endothelial, gastrointestinal and immune-cell antigens. All patients were treated with anti- IL6R antibody or IVIG, which led to rapid disease resolution tracking with normalization of inflammatory markers.

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