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1.
Trends Pharmacol Sci ; 2022 Jul 25.
Article in English | MEDLINE | ID: covidwho-1956357

ABSTRACT

Decoy receptor proteins that trick viruses to bind to them should be resistant to viral escape because viruses that require entry receptors cannot help but bind decoy receptors. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for coronavirus cell entry. Recombinant soluble ACE2 was previously developed as a biologic against acute respiratory distress syndrome (ARDS) and verified to be safe in clinical studies. The emergence of COVID-19 reignited interest in soluble ACE2 as a potential broad-spectrum decoy receptor against coronaviruses. In this review, we summarize recent developments in preclinical studies using various high-affinity mutagenesis and Fc fusion approaches to achieve therapeutic efficacy of recombinant ACE2 decoy receptor against coronaviruses. We also highlight the relevance of stimulating effector immune cells through Fc-receptor engagement and the potential of using liquid aerosol delivery of ACE2 decoy receptors for defense against ACE2-utilizing coronaviruses.

2.
Commun Biol ; 5(1): 516, 2022 05 30.
Article in English | MEDLINE | ID: covidwho-1947507

ABSTRACT

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.


Subject(s)
COVID-19 , SARS-CoV-2 , Bronchi , Humans , Organoids
3.
Commun Biol ; 5(1): 483, 2022 05 19.
Article in English | MEDLINE | ID: covidwho-1852521

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin ß1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression.


Subject(s)
COVID-19 , SARS-CoV-2 , Viral Proteins/metabolism , Animals , Antiviral Agents , Biological Transport , Cricetinae , Viral Proteins/genetics , Virus Replication , alpha Karyopherins/genetics , alpha Karyopherins/metabolism
4.
Antiviral Res ; 199: 105268, 2022 03.
Article in English | MEDLINE | ID: covidwho-1850634

ABSTRACT

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-ß but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-ß. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , High-Throughput Screening Assays , Humans , Replicon , SARS-CoV-2/genetics , Virus Replication
5.
Sci Transl Med ; 14(650): eabn7737, 2022 Jun 22.
Article in English | MEDLINE | ID: covidwho-1807308

ABSTRACT

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Animals , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/therapy , Humans , Immunization, Passive , Mice , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2
6.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-330058

ABSTRACT

Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines during worsening disease. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 generates excessive amounts of small viral RNAs (svRNAs) encoding exact 5′ ends of positive-sense genes in human cells, whereas significantly fewer similar svRNAs are produced by endemic human coronaviruses (OC43 and 229E). SARS-CoV-2 5′ end svRNAs are RIG-I agonists associated with IFN-beta expression in later stages of infection. The first 60-nt ends bearing duplex structures and 5′-triphosphates are responsible for immune-stimulation. The 5′ end svRNAs were also produced during infection ex vivo and in vivo . The delta variant retains the robust 5′ end svRNA production of the parental strain, whereas omicron (BA.1 and BA.2) produces little of these erroneous svRNAs. We propose that RIG-I activation by accumulated 5′ end svRNAs overcomes the initial IFN antagonistic ability of viral proteins and contributes to drive late over-exuberant IFN production leading to the development of severe COVID-19 and suggest that evolutionary modification of SARS-CoV-2 5′ end svRNA production may correlate with the reduced disease severity likely seen with omicron (BA.1 and BA.2).

8.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-328797

ABSTRACT

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab’) 2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19. (150 words)

9.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-328658

ABSTRACT

Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines during worsening disease 1-4 . Despite this, the interferon (IFN) response is delayed, contributing to disease progression 5 . Here, we report that SARS-CoV-2 generates excessive amounts of small viral RNAs (svRNAs) encoding exact 5′ ends of positive sense genes in human cells, whereas significantly fewer similar svRNAs are produced by endemic human coronaviruses (OC43 and 229E). SARS-CoV-2 5′ end svRNAs are potent RIG-I agonists associated with IFN-β expression in later stages of infection. The first 60-nt ends bearing duplex structures and 5′-triphosphates are responsible for immune-stimulation. The 5′ end svRNAs were also produced during infection in vivo. The delta variant retains the robust 5’ end svRNA production of the parental strain, whereas omicron no longer produces these erroneous svRNAs. We propose that RIG-I activation by accumulated 5′ end svRNAs overcomes the initial IFN antagonistic ability of viral proteins and drives late over-exuberant IFN production leading to the development of severe COVID-19 and suggest that evolutionary modification of SARS-CoV-2 5’ end svRNA production may correlate with the reduced disease severity likely seen with omicron.

10.
Nat Commun ; 12(1): 3802, 2021 06 21.
Article in English | MEDLINE | ID: covidwho-1387351

ABSTRACT

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/pharmacology , COVID-19/drug therapy , Mutation , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Cricetinae , Crystallography, X-Ray , Disease Models, Animal , Humans , Male , Molecular Dynamics Simulation , Protein Binding , Protein Engineering/methods , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism
11.
Cell ; 184(13): 3452-3466.e18, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1240207

ABSTRACT

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , COVID-19/immunology , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Protein Binding/immunology , Protein Domains/immunology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
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