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1.
J Virol ; 96(11): e0036422, 2022 Jun 08.
Article in English | MEDLINE | ID: covidwho-1854234

ABSTRACT

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made toward identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and IFN-stimulated gene (ISG) response is another antiviral strategy that has been known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the proinflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, has been shown to be less proinflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV-infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-α) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to that of IFN-α-treated cells. Similarly, mice treated with two doses of IFN-λ were better protected than mice that received a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from a lethal Middle East respiratory syndrome CoV (MERS-CoV) infection. A two-dose IFN-λ regimen significantly reduced lung viral titers and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identified an effective regimen for IFN-λ use and demonstrated the protective efficacy of IFN-λ in MERS-CoV-infected mice. IMPORTANCE Effective antiviral agents are urgently required to prevent and treat individuals infected with SARS-CoV-2 and other emerging viral infections. The COVID-19 pandemic has catapulted our efforts to identify, develop, and evaluate several antiviral agents. However, a narrow therapeutic window has limited the protective efficacy of several broad-spectrum and CoV-specific antivirals. IFN-λ is an antiviral agent of interest due to its ability to induce a robust endogenous antiviral state and low levels of inflammation. Here, we evaluated the protective efficacy and effective treatment regimen of IFN-λ in mice infected with a lethal dose of MERS-CoV. We show that while prophylactic and early therapeutic IFN-λ administration is protective, delayed treatment is detrimental. Notably, a combination of prophylactic and delayed therapeutic administration of IFN-λ protected mice from severe MERS. Our results highlight the prophylactic and therapeutic use of IFN-λ against lethal hCoV and likely other viral lung infections.


Subject(s)
Antiviral Agents , Coronavirus Infections , Interferons , Middle East Respiratory Syndrome Coronavirus , Animals , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Humans , Interferons/pharmacology , Mice
2.
Sci Rep ; 12(1): 6972, 2022 04 28.
Article in English | MEDLINE | ID: covidwho-1815600

ABSTRACT

Common alphacoronaviruses and human rhinoviruses (HRV) induce type I and III interferon (IFN) responses important to limiting viral replication in the airway epithelium. In contrast, highly pathogenic betacoronaviruses including SARS-CoV-2 may evade or antagonize RNA-induced IFN I/III responses. In airway epithelial cells (AECs) from children and older adults we compared IFN I/III responses to SARS-CoV-2 and HRV-16, and assessed whether pre-infection with HRV-16, or pretreatment with recombinant IFN-ß or IFN-λ, modified SARS-CoV-2 replication. Bronchial AECs from children (ages 6-18 years) and older adults (ages 60-75 years) were differentiated ex vivo to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 or HRV-16, and RNA and protein was harvested from cell lysates 96 h. following infection and supernatant was collected 48 and 96 h. following infection. In additional experiments cultures were pre-infected with HRV-16, or pre-treated with recombinant IFN-ß1 or IFN-λ2 before SARS-CoV-2 infection. In a subset of experiments a range of infectious concentrations of HRV-16, SARS-CoV-2 WA-01, SARS-CoV-2 Delta variant, and SARS-CoV-2 Omicron variant were studied. Despite significant between-donor heterogeneity SARS-CoV-2 replicated 100 times more efficiently than HRV-16. IFNB1, INFL2, and CXCL10 gene expression and protein production following HRV-16 infection was significantly greater than following SARS-CoV-2. IFN gene expression and protein production were inversely correlated with SARS-CoV-2 replication. Treatment of cultures with recombinant IFNß1 or IFNλ2, or pre-infection of cultures with HRV-16, markedly reduced SARS-CoV-2 replication. In addition to marked between-donor heterogeneity in IFN responses and viral replication, SARS-CoV-2 (WA-01, Delta, and Omicron variants) elicits a less robust IFN response in primary AEC cultures than does rhinovirus, and heterologous rhinovirus infection, or treatment with recombinant IFN-ß1 or IFN-λ2, reduces SARS-CoV-2 replication, although to a lesser degree for the Delta and Omicron variants.


Subject(s)
COVID-19 , Interferons , Adolescent , Aged , Antiviral Agents , COVID-19/drug therapy , Child , Humans , Interferons/pharmacology , Middle Aged , RNA , Rhinovirus , SARS-CoV-2
3.
PLoS One ; 17(4): e0266412, 2022.
Article in English | MEDLINE | ID: covidwho-1793503

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.


Subject(s)
COVID-19 , Respiratory Syncytial Virus Infections , Antiviral Agents/pharmacology , Epithelial Cells/metabolism , Humans , Interferons/metabolism , Interferons/pharmacology , Janus Kinases/metabolism , SARS-CoV-2 , STAT Transcription Factors/metabolism , Signal Transduction
4.
J Virol ; 96(7): e0170521, 2022 04 13.
Article in English | MEDLINE | ID: covidwho-1736024

ABSTRACT

The coronavirus SARS-CoV-2 caused the COVID-19 global pandemic leading to 5.3 million deaths worldwide as of December 2021. The human intestine was found to be a major viral target which could have a strong impact on virus spread and pathogenesis since it is one of the largest organs. While type I interferons (IFNs) are key cytokines acting against systemic virus spread, in the human intestine type III IFNs play a major role by restricting virus infection and dissemination without disturbing homeostasis. Recent studies showed that both type I and III IFNs can inhibit SARS-CoV-2 infection, but it is not clear whether one IFN controls SARS-CoV-2 infection of the human intestine better or with a faster kinetics. In this study, we could show that type I and III IFNs both possess antiviral activity against SARS-CoV-2 in human intestinal epithelial cells (hIECs); however, type III IFN is more potent. Shorter type III IFN pretreatment times and lower concentrations were required to efficiently reduce virus load compared to type I IFNs. Moreover, type III IFNs significantly inhibited SARS-CoV-2 even 4 h postinfection and induced a long-lasting antiviral effect in hIECs. Importantly, the sensitivity of SARS-CoV-2 to type III IFNs was virus specific since type III IFN did not control VSV infection as efficiently. Together, these results suggest that type III IFNs have a higher potential for IFN-based treatment of SARS-CoV-2 intestinal infection compared to type I IFNs. IMPORTANCE SARS-CoV-2 infection is not restricted to the respiratory tract and a large number of COVID-19 patients experience gastrointestinal distress. Interferons are key molecules produced by the cell to combat virus infection. Here, we evaluated how two types of interferons (type I and III) can combat SARS-CoV-2 infection of human gut cells. We found that type III interferons were crucial to control SARS-CoV-2 infection when added both before and after infection. Importantly, type III interferons were also able to produce a long-lasting effect, as cells were protected from SARS-CoV-2 infection up to 72 h posttreatment. This study suggested an alternative treatment possibility for SARS-CoV-2 infection.


Subject(s)
COVID-19 , Interferon Type I , Interferons , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cells, Cultured , Epithelial Cells , Humans , Interferon Type I/pharmacology , Interferons/pharmacology , SARS-CoV-2/drug effects
5.
Nat Commun ; 13(1): 679, 2022 02 03.
Article in English | MEDLINE | ID: covidwho-1671560

ABSTRACT

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.


Subject(s)
COVID-19/immunology , Interferons/pharmacology , Myeloid Cells/immunology , SARS-CoV-2/drug effects , Animals , Antiviral Agents , Bronchoalveolar Lavage , Disease Models, Animal , Humans , Immunity, Innate , Inflammation , Interferon Type I/genetics , Interferon Type I/pharmacology , Interferons/genetics , Lung/immunology , Lung/pathology , Macaca mulatta , Macrophages/immunology , T-Lymphocytes/immunology
6.
Viruses ; 14(1)2021 12 29.
Article in English | MEDLINE | ID: covidwho-1639272

ABSTRACT

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-ß response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1-), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1- cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.


Subject(s)
Diploidy , Interferons/pharmacology , Rabies Vaccines/immunology , Rabies virus , Rabies/prevention & control , Animals , Antibodies, Viral , Cell Line , Chlorocebus aethiops , Gene Expression , Humans , Interferons/genetics , Receptor, Interferon alpha-beta/genetics , Vaccines, Inactivated/immunology , Vero Cells
7.
Inflammation ; 45(3): 1348-1361, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1637685

ABSTRACT

The oral mucosa is one of the first lines of the innate host defense system against microbial invasion. Interferon (IFN) lambda-1 (IFN-λ1), a type III IFN, exhibits type I IFN-like antiviral activity. In contrast to ubiquitously expressed type I IFN receptors, IFN-λ receptor 1 (IFN-λR1), which has higher affinity for type III IFNs than low-affinity interleukin (IL)-10 receptor 2, is mainly expressed on epithelial cells. Although IFN-λ1 has been shown to exert antiviral effects in the respiratory tract, gastrointestinal tract, and skin, the regulation of type III IFN receptor expression and its functions in the oral mucosa remain unclear. We herein showed the expression of IFN-λR1 in human gingival keratinocytes. The expression of IL-6, angiotensin-converting enzyme 2 (a critical molecule for severe acute respiratory syndrome coronavirus 2 infection), and IL-8 in human primary gingival keratinocytes (HGK) were significantly higher following treatments with either type I IFN (IFN-ß) or type II IFN (IFN-γ) than with IFN-λ1. However, the IFN-λ1 treatment strongly induced toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I), which mainly recognize viral nucleic acids, via the STAT1-mediated pathway. Furthermore, a stimulation with a RIG-I or TLR3 agonist promoted the production of IL-6, IL-8, and IFN-λ in HGK, which was significantly enhanced by a pretreatment with IFN-λ1. These results suggest that IFN-λ1 may contribute to the activation of innate immune responses to oral viral infections by up-regulating the expression of RIG-I and TLR3 and priming their functions in keratinocytes.


Subject(s)
Interferons , /immunology , DEAD Box Protein 58/metabolism , Humans , Immunity, Innate , Interferons/immunology , Interferons/pharmacology , Interleukin-6 , Interleukin-8 , Mouth Mucosa/metabolism , Receptors, Immunologic/metabolism , Toll-Like Receptor 3/metabolism
8.
Bull Exp Biol Med ; 172(1): 53-56, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1520385

ABSTRACT

The antiviral activity of recombinant human IFN-lambda type 1 (IFNλ-1) against culture strain of SARS-CoV-2 virus was determined by infecting a highly sensitive VeroE6 coronavirus cell culture after preincubation test (the cell monolayer was incubated with 4-fold dilutions of IFNλ-1 in a concentration range of 0.16-42,500 ng/ml in a culture medium for 12 h at 37°C) and without preincubation (simultaneous addition of different concentrations of IFNλ-1 and SARS-CoV-2 infection in a dose of 102 TCID50). The created recombinant human IFNλ-1 demonstrated obvious antiviral activity against SARS-CoV-2 virus in vitro. In the tests with and without preincubation, IFNλ-1 exhibited significant activity, although somewhat lower in variant with simultaneous addition of IFNλ-1 and virus to the cell culture. It should be noted that the antiviral effect of IFNλ-1 was observed in a wide range of concentrations.


Subject(s)
Antiviral Agents/pharmacology , Interferons/pharmacology , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , Viral Load/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/isolation & purification , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Interferons/biosynthesis , Interferons/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Vero Cells , Viral Load/genetics
9.
mBio ; 12(6): e0275621, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1494976

ABSTRACT

Outbreaks of emerging viral pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a major medical challenge. There is a pressing need for antivirals that can be rapidly deployed to curb infection and dissemination. We determined the efficacy of interferon lambda-1 (IFN-λ) as a broad-spectrum antiviral agent to inhibit SARS-CoV-2 infection and reduce pathology in a mouse model of disease. IFN-λ significantly limited SARS-CoV-2 production in primary human bronchial epithelial cells in culture. Pretreatment of human lung cells with IFN-λ completely blocked infectious virus production, and treatment with IFN-λ at the time of infection inhibited virus production more than 10-fold. To interrogate the protective effects of IFN-λ in response to SARS-CoV-2 infection, transgenic mice expressing the human angiotensin-converting enzyme 2 (ACE-2) were tested. One dose of IFN-λ administered intranasally was found to reduce animal morbidity and mortality. Our study with SARS-CoV-2 also revealed a sex differential in disease outcome. Male mice had higher mortality, reflecting the more severe symptoms and mortality found in male patients infected with SARS-CoV-2. The results indicate that IFN-λ potentially can treat early stages of SARS-CoV-2 infection and decrease pathology, and this murine model can be used to investigate the sex differential documented in COVID-19. IMPORTANCE The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease. Specific vaccines have been responsible for curbing the transmission of SARS-CoV-2 in developed nations. However, vaccines require time to generate and keep pace with antigenic variants. There is a need for broad-spectrum prophylactic and therapeutic agents to combat new emerging viral pathogens. Our mouse model suggests IFN-λ has clinical utility, and it reflects the well-documented finding that male COVID-19 patients manifest more severe symptoms and mortality. Understanding this sex bias is critical for considering therapeutic approaches to COVID-19.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/immunology , COVID-19/therapy , Epithelial Cells/drug effects , Interferons/immunology , Interferons/pharmacology , SARS-CoV-2/immunology , Administration, Intranasal , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/pharmacology , Bronchi/cytology , Disease Models, Animal , Epithelial Cells/immunology , Epithelial Cells/virology , Female , HEK293 Cells , Humans , Interferons/classification , Lung/drug effects , Lung/pathology , Lung/virology , Male , Mice , Mice, Transgenic , Risk Factors , SARS-CoV-2/drug effects , Sex Factors
10.
Science ; 374(6571): 1099-1106, 2021 Nov 26.
Article in English | MEDLINE | ID: covidwho-1467657

ABSTRACT

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.


Subject(s)
RNA, Viral/genetics , Replicon/physiology , SARS-CoV-2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , Cell Line , Humans , Interferons/pharmacology , Microbial Sensitivity Tests , Mutation , Plasmids , RNA, Viral/metabolism , Replicon/genetics , Reverse Genetics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virion/genetics , Virion/physiology , Virus Replication
11.
ACS Chem Biol ; 16(5): 844-856, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1457790

ABSTRACT

Interferon-induced transmembrane proteins (IFITMs) are S-palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S-palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how S-palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific S-palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific S-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.


Subject(s)
Antiviral Agents/chemistry , Cell Membrane/metabolism , Interferons/chemistry , Lipids/chemistry , Membrane Proteins/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites , Cell Membrane/ultrastructure , Computational Biology , Drug Design , Humans , Interferons/pharmacology , Lipoylation , Lysosomes/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Signal Transduction
12.
Viruses ; 13(8)2021 08 12.
Article in English | MEDLINE | ID: covidwho-1436097

ABSTRACT

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.


Subject(s)
Adenoviruses, Human/drug effects , Chikungunya virus/drug effects , Influenza A virus/drug effects , Interferons/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adenoviruses, Human/physiology , Animals , Chikungunya virus/physiology , Chlorocebus aethiops , Dose-Response Relationship, Drug , Gene Expression Regulation , Humans , Influenza A virus/physiology , Interferons/therapeutic use , Interleukins , RNA Virus Infections/drug therapy , RNA Virus Infections/prevention & control , Recombinant Proteins/pharmacology , SARS-CoV-2/physiology , Vero Cells , Viral Load/drug effects , Virus Replication/drug effects
13.
Cytokine ; 140: 155430, 2021 04.
Article in English | MEDLINE | ID: covidwho-1385381

ABSTRACT

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS-CoV-2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs' activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs' activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS-CoV-2 infection in the respiratory tract.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/prevention & control , Epithelial Cells/drug effects , Gene Expression/drug effects , Interferons/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adult , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/virology , Cell Line, Tumor , Child , Cytokines/genetics , Epithelial Cells/metabolism , Humans , Interferons/metabolism , Lung/cytology , Middle Aged , SARS-CoV-2/physiology , Ubiquitins/genetics
14.
STAR Protoc ; 2(4): 100781, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1356489

ABSTRACT

We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli. For complete details on the use and execution of this protocol, please refer to Hayn et al. (2021).


Subject(s)
COVID-19/pathology , Gene Expression Regulation, Viral/drug effects , Interferons/pharmacology , Luciferases/metabolism , RNA, Messenger/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Humans , Luciferases/genetics , Promoter Regions, Genetic , RNA, Messenger/genetics , SARS-CoV-2/drug effects , Viral Proteins/genetics
15.
Ann Clin Transl Neurol ; 8(8): 1738-1744, 2021 08.
Article in English | MEDLINE | ID: covidwho-1300348

ABSTRACT

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39-3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18-0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , COVID-19/physiopathology , Immunologic Factors/pharmacology , Interferons/pharmacology , Multiple Sclerosis/drug therapy , Rituximab/pharmacology , Adult , Antibodies, Monoclonal, Humanized/adverse effects , COVID-19/epidemiology , Female , France/epidemiology , Humans , Immunologic Factors/adverse effects , Interferons/adverse effects , Italy/epidemiology , Male , Meta-Analysis as Topic , Middle Aged , Multiple Sclerosis/epidemiology , Multivariate Analysis , Protective Factors , Retrospective Studies , Risk Factors , Rituximab/adverse effects , Severity of Illness Index
16.
Commun Biol ; 4(1): 654, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1253994

ABSTRACT

SARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Here, we report on transcriptomes activated in primary airway cells by interferons and their suppression by Janus kinase (JAK) inhibitors. Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. ChIP-seq for activating histone marks and Pol II loading identified candidate enhancer elements controlling the ACE2 locus, including the intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE2 and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib, used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/pharmacology , COVID-19/drug therapy , Interferons/pharmacology , Janus Kinase Inhibitors/pharmacology , Transcriptional Activation/drug effects , COVID-19/genetics , Cell Line , Humans , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Transcriptome/drug effects
17.
PLoS Pathog ; 17(5): e1009229, 2021 05.
Article in English | MEDLINE | ID: covidwho-1239922

ABSTRACT

While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.


Subject(s)
Camelids, New World , Coronavirus Infections/immunology , Interferon Type I/metabolism , Interferons/metabolism , Middle East Respiratory Syndrome Coronavirus/immunology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Camelids, New World/immunology , Camelids, New World/metabolism , Camelids, New World/virology , Chlorocebus aethiops , Coronavirus Infections/metabolism , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Disease Reservoirs/veterinary , Disease Resistance/drug effects , Disease Resistance/genetics , Disease Resistance/immunology , Gene Expression Regulation , Immunity, Innate/physiology , Inflammation/immunology , Inflammation/metabolism , Inflammation/veterinary , Inflammation/virology , Interferon Type I/genetics , Interferon Type I/pharmacology , Interferons/genetics , Interferons/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , Nasal Mucosa/drug effects , Nasal Mucosa/immunology , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Respiratory System/drug effects , Respiratory System/immunology , Respiratory System/metabolism , Respiratory System/virology , Vero Cells , Viral Load/drug effects , Virus Replication/drug effects
18.
Food Chem Toxicol ; 150: 112087, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1188569

ABSTRACT

Coronavirus disease-19 (COVID-19) is a complex disease that causes illness ranging from mild to severe respiratory problems. It is caused by a novel coronavirus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) that is an enveloped positive-sense single-stranded RNA (+ssRNA) virus belongs to coronavirus CoV family. It has a fast-spreading potential worldwide, which leads to high mortality regardless of lows death rates. Now some vaccines or a specific drug are approved but not available for every country for disease prevention and/or treatment. Therefore, it is a high demand to identify the known drugs and test them as a possible therapeutic approach. In this critical situation, one or more of these drugs may represent the only option to treat or reduce the severity of the disease, until some specific drugs or vaccines will be developed and/or approved for everyone in this pandemic. In this updated review, the available repurpose immunotherapeutic treatment strategies are highlighted, elucidating the crosstalk between the immune system and SARS-CoV-2. Despite the reasonable data availability, the effectiveness and safety of these drugs against SARS-CoV-2 needs further studies and validations aiming for a better clinical outcome.


Subject(s)
Antiviral Agents/pharmacology , Inflammation/etiology , SARS-CoV-2/drug effects , Adrenal Cortex Hormones/pharmacology , Adrenal Cortex Hormones/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/immunology , COVID-19/virology , Cytokines/metabolism , Humans , Inflammation/drug therapy , Inflammation/immunology , Interferons/pharmacology , Interferons/therapeutic use , Janus Kinase Inhibitors/pharmacology , Janus Kinase Inhibitors/therapeutic use , SARS-CoV-2/immunology
19.
PLoS Biol ; 19(3): e3001158, 2021 03.
Article in English | MEDLINE | ID: covidwho-1156073

ABSTRACT

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.


Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation, Viral/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , Antiviral Agents/pharmacology , Cells, Cultured , Chlorocebus aethiops , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression Regulation, Viral/drug effects , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Interferons/pharmacology , SARS Virus/drug effects , SARS Virus/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Species Specificity , Temperature , Vero Cells , Virus Replication/drug effects , Virus Replication/genetics
20.
mBio ; 12(2)2021 03 16.
Article in English | MEDLINE | ID: covidwho-1138303

ABSTRACT

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.


Subject(s)
Membrane Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Cells, Cultured , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Interferons/pharmacology , Intracellular Space/metabolism , Membrane Proteins/genetics , Mutation , RNA Viruses/classification , RNA Viruses/metabolism , Species Specificity , Ubiquitin-Protein Ligases/genetics , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Virion/metabolism , Virus Replication
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