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1.
Sci Rep ; 12(1): 10340, 2022 Jun 20.
Article in English | MEDLINE | ID: covidwho-1900653

ABSTRACT

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Saudi Arabia and was mostly associated with severe respiratory illness in humans. Dromedary camels are the zoonotic reservoir for MERS-CoV. To investigate the biology of MERS-CoV in camelids, we developed a well-differentiated airway epithelial cell (AEC) culture model for Llama glama and Camelus bactrianus. Histological characterization revealed progressive epithelial cellular differentiation with well-resemblance to autologous ex vivo tissues. We demonstrate that MERS-CoV displays a divergent cell tropism and replication kinetics profile in both AEC models. Furthermore, we observed that in the camelid AEC models MERS-CoV replication can be inhibited by both type I and III interferons (IFNs). In conclusion, we successfully established camelid AEC cultures that recapitulate the in vivo airway epithelium and reflect MERS-CoV infection in vivo. In combination with human AEC cultures, this system allows detailed characterization of the molecular basis of MERS-CoV cross-species transmission in respiratory epithelium.


Subject(s)
Camelids, New World , Coronavirus Infections , Middle East Respiratory Syndrome Coronavirus , Animals , Camelus , Respiratory System
2.
J Virol ; 96(11): e0036422, 2022 06 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
3.
J Interferon Cytokine Res ; 41(11): 407-414, 2021 11.
Article in English | MEDLINE | ID: covidwho-1758604

ABSTRACT

Genetic polymorphisms at the IFNL4 loci are known to influence the clinical outcome of several different infectious diseases. Best described is the association between the IFNL4 genotype and hepatitis C virus clearance. However, an influence of the IFNL4 genotype on the adaptive immune system was suggested by several studies but never investigated in humans. In this cross-sectional study, we have genotyped 201 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive participants for 3 IFNL4 polymorphisms (rs368234815, rs12979860, and rs117648444) and stratified them according to the IFNλ4 activity. Based on this stratification, we investigated the association between the IFNL4 genotype and the antibody as well as the CD8+ T cell response in the acute phase of the SARS-CoV-2 infection. We observed no differences in the genotype distribution compared with a Danish reference cohort or the 1,000 Genome Project, and we were not able to link the IFNL4 genotype to changes in either the antibody or CD8+ T cell responses of these patients.


Subject(s)
Adaptive Immunity/immunology , COVID-19/immunology , Interleukins/immunology , SARS-CoV-2/immunology , Adaptive Immunity/genetics , Adult , Aged , CD8-Positive T-Lymphocytes/immunology , Cohort Studies , Cross-Sectional Studies , Female , Genotype , Humans , Interleukins/genetics , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Polymorphism, Single Nucleotide/immunology , SARS-CoV-2/genetics , Young Adult
6.
PLoS Pathog ; 17(8): e1009800, 2021 08.
Article in English | MEDLINE | ID: covidwho-1435629

ABSTRACT

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.


Subject(s)
Interferon-beta/metabolism , SARS-CoV-2/immunology , Viral Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Chlorocebus aethiops , Eukaryotic Initiation Factor-2/metabolism , HEK293 Cells , Humans , Interferon-beta/genetics , Interferon-beta/pharmacology , SARS-CoV-2/drug effects , STAT1 Transcription Factor/metabolism , Vero Cells , Viral Proteins/genetics
7.
EBioMedicine ; 68: 103410, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1252688

ABSTRACT

BACKGROUND: The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search and evaluation of new treatment modalities and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. METHODS: We included 203 recovered SARS-CoV-2 infected patients in Denmark between April 3rd and July 9th 2020, at least 14 days after COVID-19 symptom recovery. The participants had experienced a range of disease severities from asymptomatic to severe. We collected plasma, serum and PBMC's for analysis of SARS-CoV-2 specific antibody response by Meso Scale analysis including other coronavirus strains, ACE2 competition, IgA ELISA, pseudovirus neutralization capacity, and dextramer flow cytometry analysis of CD8+ T cells. The immunological outcomes were compared amongst severity groups within the cohort, and 10 pre-pandemic SARS-CoV-2 negative controls. FINDINGS: We report broad serological profiles within the cohort, detecting antibody binding to other human coronaviruses. 202(>99%) participants had SARS-CoV-2 specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 193(95%) individuals. A significant positive correlation (r=0.7804) between spike-ACE2 blocking antibody titers and neutralization potency was observed. Further, SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 95 of 106(90%) HLA-A2+ individuals. INTERPRETATION: The viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T-cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of their disease severity. FUNDING: This study was supported by the Danish Ministry for Research and Education (grant# 0238-00001B) and The Danish Innovation Fund (grant# 0208-00018B).


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/blood , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adaptive Immunity , Adult , Aged , Animals , Antibodies, Viral/blood , CD8-Positive T-Lymphocytes/metabolism , COVID-19/virology , Cell Line , Denmark , Female , Humans , Male , Middle Aged , SARS-CoV-2/pathogenicity , Severity of Illness Index , Young Adult
8.
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
9.
EMBO Rep ; 21(12): e51252, 2020 12 03.
Article in English | MEDLINE | ID: covidwho-895751

ABSTRACT

Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV-2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of pro-inflammatory cytokines later during infection.


Subject(s)
COVID-19/immunology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/virology , SARS-CoV-2/immunology , Antiviral Agents/immunology , COVID-19/virology , Cells, Cultured , Cytokines/immunology , Epithelial Cells/immunology , Epithelial Cells/virology , Humans , Immune Evasion , Interferon Type I/immunology , Lung/immunology , Lung/virology , Pandemics
11.
J Biol Chem ; 295(41): 13958-13964, 2020 10 09.
Article in English | MEDLINE | ID: covidwho-615996

ABSTRACT

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS-CoV-2 and compared them with those against SARS-CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS-CoV-2. In contrast, SARS-CoV-1 was restricted only by IFN-α in these cell lines. SARS-CoV-2 generally exhibited a broader IFN sensitivity than SARS-CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS-CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS-CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect-prone type III IFN are good candidates for the management of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Interferon Type I/pharmacology , Interferons/pharmacology , Animals , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , COVID-19 , Cell Line , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Janus Kinases/metabolism , Nitriles , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Pyrazoles/pharmacology , Pyrimidines , SARS Virus/drug effects , SARS Virus/physiology , SARS-CoV-2 , Signal Transduction/drug effects , Vero Cells , Virus Replication/drug effects
12.
Science ; 369(6504): 712-717, 2020 08 07.
Article in English | MEDLINE | ID: covidwho-594812

ABSTRACT

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-ß) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-ß responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-λ driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19).


Subject(s)
Alveolar Epithelial Cells/pathology , Cytokines/metabolism , Interferon Type I/metabolism , Interferons/metabolism , Lung/pathology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/pathology , Alveolar Epithelial Cells/immunology , Animals , Apoptosis , Bronchoalveolar Lavage Fluid/immunology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cytokines/administration & dosage , Cytokines/immunology , Female , Influenza A Virus, H3N2 Subtype , Interferon Type I/administration & dosage , Interferon Type I/pharmacology , Interferon-alpha/administration & dosage , Interferon-alpha/metabolism , Interferon-alpha/pharmacology , Interferon-beta/administration & dosage , Interferon-beta/metabolism , Interferon-beta/pharmacology , Interferons/administration & dosage , Interferons/pharmacology , Male , Mice , Orthomyxoviridae Infections/metabolism , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Receptors, Interferon/genetics , Receptors, Interferon/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolism
13.
J Exp Med ; 217(5)2020 05 04.
Article in English | MEDLINE | ID: covidwho-60343

ABSTRACT

With the first reports on coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the scientific community working in the field of type III IFNs (IFN-λ) realized that this class of IFNs could play an important role in this and other emerging viral infections. In this Viewpoint, we present our opinion on the benefits and potential limitations of using IFN-λ to prevent, limit, and treat these dangerous viral infections.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/metabolism , Interferons/metabolism , Pneumonia, Viral/metabolism , COVID-19 , Humans , Pandemics , SARS-CoV-2 , Virus Internalization
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