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1.
Nature ; 601(7891): 110-117, 2022 01.
Article in English | MEDLINE | ID: covidwho-1510600

ABSTRACT

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-3. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4-11), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)12,13, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref. 14), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae.


Subject(s)
Asymptomatic Infections , COVID-19/immunology , COVID-19/virology , DNA-Directed RNA Polymerases/immunology , SARS-CoV-2/immunology , Seroconversion , Cell Proliferation , Cohort Studies , DNA-Directed RNA Polymerases/metabolism , Evolution, Molecular , Female , Health Personnel , Humans , Male , Membrane Proteins/immunology , Multienzyme Complexes/immunology , SARS-CoV-2/enzymology , SARS-CoV-2/growth & development , Transcription, Genetic/immunology
2.
Viruses ; 13(11)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1502526

ABSTRACT

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the "pairing potential" of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the "inside" or "outside" of the infected cell. Their therapeutic potential will be discussed.


Subject(s)
Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Viroporin Proteins/immunology , Animals , Biomarkers , COS Cells , Cell Surface Display Techniques/methods , Chlorocebus aethiops , Epitopes/immunology , HEK293 Cells , Humans , Membrane Proteins/immunology , Protein Domains , Vero Cells
3.
Adv Drug Deliv Rev ; 179: 114020, 2021 12.
Article in English | MEDLINE | ID: covidwho-1486938

ABSTRACT

Adjuvant is an essential component in subunit vaccines. Many agonists of pathogen recognition receptors have been developed as potent adjuvants to optimize the immunogenicity and efficacy of vaccines. Recently discovered cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway has attracted much attention as it is a key mediator for modulating immune responses. Vaccines adjuvanted with STING agonists are found to mediate a robust immune defense against infections and cancer. In this review, we first discuss the mechanisms of STING agonists in the context of vaccination. Next, we present recent progress in novel STING agonist discovery and the delivery strategies. We next highlight recent work in optimizing the efficacy while minimizing toxicity of STING agonist-assisted subunit vaccines for protection against infectious diseases or treatment of cancer. Finally, we share our perspectives of current issues and future directions in further developing STING agonists for adjuvanting subunit vaccines.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Membrane Proteins/agonists , Membrane Proteins/immunology , Vaccines, Subunit/immunology , CD4-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/drug effects , Dendritic Cells/drug effects , Humans , Immunity, Humoral/drug effects , Nucleotidyltransferases/metabolism
4.
Cell Mol Life Sci ; 78(23): 7427-7434, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1491059

ABSTRACT

Viral infections pose a severe threat to humans by causing many infectious, even fatal, diseases, such as the current pandemic disease (COVID-19) since 2019, and understanding how the host innate immune system recognizes viruses has become more important. Endosomal and cytosolic sensors can detect viral nucleic acids to induce type I interferon and proinflammatory cytokines, subsequently inducing interferon-stimulated genes for restricting viral infection. Although viral RNA and DNA sensing generally rely on diverse receptors and adaptors, the crosstalk between DNA and RNA sensing is gradually appreciated. This minireview highlights the overlap between the RNA- and DNA-sensing mechanisms in antiviral innate immunity, which significantly amplifies the antiviral innate responses to restrict viral infection and might be a potential novel target for preventing and treating viral diseases.


Subject(s)
COVID-19/immunology , DNA, Viral/immunology , Immunity, Innate/immunology , RNA, Viral/immunology , SARS-CoV-2/immunology , COVID-19/prevention & control , Cytokines/metabolism , Endosomes/immunology , Humans , Interferon Type I/metabolism , Membrane Proteins/immunology , Nuclear Proteins/immunology , Phosphoproteins/immunology
5.
J Med Virol ; 93(9): 5376-5389, 2021 09.
Article in English | MEDLINE | ID: covidwho-1363676

ABSTRACT

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.


Subject(s)
DEAD Box Protein 58/immunology , Immune Evasion/genetics , Interferons/immunology , Nucleotidyltransferases/immunology , Receptors, Immunologic/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 3/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Animals , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/genetics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferons/genetics , Membrane Proteins/genetics , Membrane Proteins/immunology , Nucleotidyltransferases/genetics , Phosphoproteins/genetics , Phosphoproteins/immunology , Plasmids/chemistry , Plasmids/metabolism , /immunology , Receptors, Immunologic/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 3/genetics , Transfection , Vero Cells , Virus Replication/immunology
6.
Commun Biol ; 4(1): 366, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1351981

ABSTRACT

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.


Subject(s)
Chromatography, Gel , Green Fluorescent Proteins/metabolism , Membrane Proteins/metabolism , Nanotechnology , Peptides/metabolism , Single-Domain Antibodies/immunology , Animals , Cryoelectron Microscopy , Cysteine Loop Ligand-Gated Ion Channel Receptors/genetics , Cysteine Loop Ligand-Gated Ion Channel Receptors/immunology , Cysteine Loop Ligand-Gated Ion Channel Receptors/metabolism , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Oryzias/genetics , Oryzias/metabolism , Peptides/genetics , Peptides/immunology , Protein Stability , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spectrometry, Fluorescence , Temperature , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism
7.
Viruses ; 13(7)2021 06 30.
Article in English | MEDLINE | ID: covidwho-1287278

ABSTRACT

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.


Subject(s)
Host-Pathogen Interactions , Membrane Proteins/genetics , Viral Envelope Proteins , Virion/metabolism , Viruses/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Leukemia Virus, Murine/metabolism , Membrane Proteins/immunology , Retroviridae/classification , Retroviridae/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Virion/genetics , Virus Internalization , Viruses/chemistry , Viruses/classification , Viruses/genetics
8.
J Mol Biol ; 433(20): 167093, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1260799

ABSTRACT

The announcement of the outstanding performance of AlphaFold 2 in the CASP 14 protein structure prediction competition came at the end of a long year defined by the COVID-19 pandemic. With an infectious organism dominating the world stage, the developers of Alphafold 2 were keen to play their part, accurately predicting novel structures of two proteins from SARS-CoV-2. In their blog post of December 2020, they highlighted this contribution, writing "we've also seen signs that protein structure prediction could be useful in future pandemic response efforts". So, what role does structural biology play in guiding vaccine immunogen design and what might be the contribution of AlphaFold 2?


Subject(s)
Pandemics/prevention & control , Software , Vaccines/chemistry , Antibodies, Neutralizing/immunology , Drug Design , Epitopes/chemistry , Humans , Membrane Proteins/chemistry , Membrane Proteins/immunology , Protein Conformation
9.
J Immunol ; 206(10): 2420-2429, 2021 05 15.
Article in English | MEDLINE | ID: covidwho-1215526

ABSTRACT

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.


Subject(s)
COVID-19/immunology , GTP-Binding Proteins/immunology , Immunity, Innate , Interferon Regulatory Factor-3/immunology , Membrane Proteins/immunology , SARS-CoV-2/immunology , Signal Transduction/immunology , Transglutaminases/immunology , Animals , COVID-19/genetics , COVID-19/pathology , GTP-Binding Proteins/genetics , Humans , Interferon Regulatory Factor-3/genetics , Interferon-beta/genetics , Interferon-beta/immunology , Membrane Proteins/genetics , Mice , Mice, Knockout , Signal Transduction/genetics , Transglutaminases/genetics
10.
J Med Virol ; 93(9): 5376-5389, 2021 09.
Article in English | MEDLINE | ID: covidwho-1206842

ABSTRACT

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.


Subject(s)
DEAD Box Protein 58/immunology , Immune Evasion/genetics , Interferons/immunology , Nucleotidyltransferases/immunology , Receptors, Immunologic/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 3/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Animals , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/genetics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferons/genetics , Membrane Proteins/genetics , Membrane Proteins/immunology , Nucleotidyltransferases/genetics , Phosphoproteins/genetics , Phosphoproteins/immunology , Plasmids/chemistry , Plasmids/metabolism , /immunology , Receptors, Immunologic/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 3/genetics , Transfection , Vero Cells , Virus Replication/immunology
11.
Signal Transduct Target Ther ; 6(1): 123, 2021 03 15.
Article in English | MEDLINE | ID: covidwho-1135650

ABSTRACT

The emergence of SARS-CoV-2 has resulted in the COVID-19 pandemic, leading to millions of infections and hundreds of thousands of human deaths. The efficient replication and population spread of SARS-CoV-2 indicates an effective evasion of human innate immune responses, although the viral proteins responsible for this immune evasion are not clear. In this study, we identified SARS-CoV-2 structural proteins, accessory proteins, and the main viral protease as potent inhibitors of host innate immune responses of distinct pathways. In particular, the main viral protease was a potent inhibitor of both the RLR and cGAS-STING pathways. Viral accessory protein ORF3a had the unique ability to inhibit STING, but not the RLR response. On the other hand, structural protein N was a unique RLR inhibitor. ORF3a bound STING in a unique fashion and blocked the nuclear accumulation of p65 to inhibit nuclear factor-κB signaling. 3CL of SARS-CoV-2 inhibited K63-ubiquitin modification of STING to disrupt the assembly of the STING functional complex and downstream signaling. Diverse vertebrate STINGs, including those from humans, mice, and chickens, could be inhibited by ORF3a and 3CL of SARS-CoV-2. The existence of more effective innate immune suppressors in pathogenic coronaviruses may allow them to replicate more efficiently in vivo. Since evasion of host innate immune responses is essential for the survival of all viruses, our study provides insights into the design of therapeutic agents against SARS-CoV-2.


Subject(s)
Immunity, Innate , Membrane Proteins/immunology , Nucleotidyltransferases/immunology , RNA, Viral/immunology , SARS-CoV-2/immunology , Signal Transduction/immunology , Viral Proteins/immunology , A549 Cells , Animals , Chickens , HEK293 Cells , HeLa Cells , Humans , Ligases/immunology , Mice
13.
Front Immunol ; 11: 598402, 2020.
Article in English | MEDLINE | ID: covidwho-1045523

ABSTRACT

COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive cellular therapy using donor-derived virus-specific T cells. One approach is the establishment of banks of HLA-typed virus-specific T cells for rapid deployment to patients. Here we show that SARS-CoV-2-exposed blood donations contain CD4 and CD8 memory T cells which recognize SARS-CoV-2 spike, nucleocapsid and membrane antigens. Peptides of these antigens can be used to isolate virus-specific T cells in a GMP-compliant process. The isolated T cells can be rapidly expanded using GMP-compliant reagents for use as an allogeneic therapy. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 1010 to 1011 T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for potential treatment of COVID-19 patients.


Subject(s)
Allogeneic Cells/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Blood Donors , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunologic Memory/immunology , Immunotherapy, Adoptive , Lymphocyte Activation/immunology , Membrane Proteins/immunology , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology
14.
Front Immunol ; 11: 607069, 2020.
Article in English | MEDLINE | ID: covidwho-993358

ABSTRACT

Upon recognition of microbial DNA or self-DNA, the cyclic-GMP-AMP synthase (cGAS) of the host catalyzes the production of the cyclic dinucleotide cGAMP. cGAMP is the main activator of STING, stimulator of interferon genes, leading to interferon synthesis through the STING-TBK1-IRF3 pathway. STING is also a hub for activation of NF-κB and autophagy. The present review details the striking similarities between T and B cell responses in severe coronavirus disease 2019 (COVID-19) and both animal or human models of STING gain of function (SAVI syndromes: STING-associated vasculopathy with onset in infancy). Those similarities may be further clues for a delayed activation of STING in severe COVID-19 patients, due to DNA damages following severe acute respiratory syndrome coronaviruses (SARS-CoV-2) infection and unusual role of STING in SARS-CoV-2 control. In early stages, Th2 differentiation are noticed in both severe COVID-19 and SAVI syndromes; then, CD4+ and CD8+ T cells functional exhaustion/senescent patterns due to TCR hyper-responsiveness are observed. T cell delayed over-responses can contribute to pneumonitis and delayed cytokine secretion with over-production of IL-6. Last, STING over-activation induces progressive CD4+ and CD8+ T lymphopenia in SAVI syndromes, which parallels what is observed in severe COVID-19. ACE2, the main receptor of SARS-CoV-2, is rarely expressed in immune cells, and it has not been yet proven that some human lymphocytes could be infected by SARS-CoV-2 through CD147 or CD26. However, STING, expressed in humans T cells, might be triggered following excessive transfer of cGAMP from infected antigen presenting cells into activated CD4+ and CD8+ T cells lymphocytes. Indeed, those lymphocytes highly express the cGAMP importer SLC19A1. Whereas STING is not expressed in human B cells, B cells counts are much less affected, either in COVID-19 or SAVI syndromes. The recognition of delayed STING over-activation in severe COVID-19 patients could prompt to target STING with specific small molecules inhibitors already designed and/or aspirin, which inhibits cGAS.


Subject(s)
B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Membrane Proteins/immunology , SARS-CoV-2/immunology , Th2 Cells/immunology , Angiotensin-Converting Enzyme 2/immunology , Animals , B-Lymphocytes/pathology , Basigin/immunology , CD8-Positive T-Lymphocytes/pathology , COVID-19/pathology , Dipeptidyl Peptidase 4/immunology , Humans , Interferon Regulatory Factor-3/immunology , Nucleotidyltransferases/immunology , Signal Transduction/immunology , Th2 Cells/pathology
15.
Chem Commun (Camb) ; 57(4): 504-507, 2021 Jan 14.
Article in English | MEDLINE | ID: covidwho-983835

ABSTRACT

A novel STING agonist, CDGSF, ipsilaterally modified with phosphorothioate and fluorine, was synthesized. The phosphorothioate in CDGSF might be a site for covalent conjugation. Injection of CDGSF generated an immunogenic ("hot") tumor microenvironment to suppress melanoma, more efficiently than dithio CDG. In particular, immunization with SARS-CoV-2 spike protein using CDGSF as an adjuvant elicited an exceptionally high antibody titer and a robust T cell response, overcoming the drawbacks of aluminum hydroxide. These results highlighted the therapeutic potential of CDGSF for cancer immunotherapy and the adjuvant potential of the STING agonist in the SARS-CoV-2 vaccine for the first time.


Subject(s)
Adjuvants, Immunologic/administration & dosage , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Melanoma, Experimental/drug therapy , Membrane Proteins/agonists , Nucleotides, Cyclic/administration & dosage , Skin Neoplasms/drug therapy , Adjuvants, Immunologic/chemical synthesis , Aluminum Hydroxide/administration & dosage , Aluminum Hydroxide/chemistry , Animals , Antibodies, Viral/biosynthesis , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/virology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/chemistry , Enzyme-Linked Immunospot Assay , Humans , Immunotherapy/methods , Interferon-gamma/biosynthesis , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Nucleotides, Cyclic/chemical synthesis , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Survival Analysis , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology , Tumor Burden/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Vaccination/methods
16.
Cells ; 9(11)2020 10 29.
Article in English | MEDLINE | ID: covidwho-902483

ABSTRACT

Cumulative data link cytokine storms with coronavirus disease 2019 (COVID-19) severity. The precise identification of immune cell subsets in bronchoalveolar lavage (BAL) and their correlation with COVID-19 disease severity are currently being unraveled. Herein, we employed iterative clustering and guide-gene selection 2 (ICGS2) as well as uniform manifold approximation and projection (UMAP) dimensionality reduction computational algorithms to decipher the complex immune and cellular composition of BAL, using publicly available datasets from a total of 68,873 single cells derived from two healthy subjects, three patients with mild COVID-19, and five patients with severe COVID-19. Our analysis revealed the presence of neutrophils and macrophage cluster-1 as a hallmark of severe COVID-19. Among the identified gene signatures, IFITM2, IFITM1, H3F3B, SAT1, and S100A8 gene signatures were highly associated with neutrophils, while CCL8, CCL3, CCL2, KLF6, and SPP1 were associated with macrophage cluster-1 in severe-COVID-19 patients. Interestingly, although macrophages were also present in healthy subjects and patients with mild COVID-19, they had different gene signatures, indicative of interstitial and cluster-0 macrophage (i.e., FABP4, APOC1, APOE, C1QB, and NURP1). Additionally, MALAT1, NEAT1, and SNGH25 were downregulated in patients with mild and severe COVID-19. Interferon signaling, FCγ receptor-mediated phagocytosis, IL17, and Tec kinase canonical pathways were enriched in patients with severe COVID-19, while PD-1 and PDL-1 pathways were suppressed. A number of upstream regulators (IFNG, PRL, TLR7, PRL, TGM2, TLR9, IL1B, TNF, NFkB, IL1A, STAT3, CCL5, and others) were also enriched in BAL cells from severe COVID-19-affected patients compared to those from patients with mild COVID-19. Further analyses revealed genes associated with the inflammatory response and chemotaxis of myeloid cells, phagocytes, and granulocytes, among the top activated functional categories in BAL from severe COVID-19-affected patients. Transcriptome data from another cohort of COVID-19-derived peripheral blood mononuclear cells (PBMCs) revealed the presence of several genes common to those found in BAL from patients with severe and mild COVID-19 (IFI27, IFITM3, IFI6, IFIT3, MX1, IFIT1, OASL, IFI30, OAS1) or to those seen only in BAL from severe-COVID-19 patients (S100A8, IFI44, IFI44L, CXCL8, CCR1, PLSCR1, EPSTI1, FPR1, OAS2, OAS3, IL1RN, TYMP, BCL2A1). Taken together, our data reveal the presence of neutrophils and macrophage cluster-1 as the main immune cell subsets associated with severe COVID-19 and identify their inflammatory and chemotactic gene signatures, also partially reflected systemically in the circulation, for possible diagnostic and therapeutic interventions.


Subject(s)
COVID-19/genetics , COVID-19/immunology , Macrophages/immunology , Neutrophils/immunology , SARS-CoV-2/immunology , Adult , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Case-Control Studies , Cohort Studies , Computational Biology/methods , Gene Expression Profiling/methods , Humans , Immunity, Innate , Interleukin-8/immunology , Membrane Proteins/immunology , SARS-CoV-2/isolation & purification , Single-Cell Analysis/methods
17.
Blood ; 136(25): 2905-2917, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-890058

ABSTRACT

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Cell Culture Techniques/methods , Immunotherapy, Adoptive/methods , SARS-CoV-2/immunology , Adult , Aged , COVID-19/drug therapy , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunodominant Epitopes/immunology , Male , Membrane Proteins/immunology , Middle Aged , Viral Proteins/immunology , Young Adult
18.
Biochimie ; 177: 50-52, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-713261

ABSTRACT

Various interferon (IFN)-inducible transmembrane (IFITM) proteins are known to be expressed in human tissues though only IFITM 1-3 are inducible by IFN. Numerous studies have shown that activation of IFITM3 could suppress infection by influenza and coronaviruses such as the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). In view of the potential application of IFITM proteins' induction to target SARS-CoV-2 infection that causes COVID-19, this article layout insights into the known antiviral mechanisms and therapeutic agents related to IFITM. Blocking viral entry through various mechanisms and the potential application of the FDA approved immunosuppressant agent, mycophenolic acid, as inducer of IFITM3 are among those discussed.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Interferons/pharmacology , Membrane Proteins/drug effects , Mycophenolic Acid/pharmacology , Pneumonia, Viral/drug therapy , RNA-Binding Proteins/drug effects , Animals , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/metabolism , Humans , Immunosuppressive Agents/pharmacology , Membrane Proteins/immunology , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/metabolism , RNA-Binding Proteins/immunology , SARS-CoV-2
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