Publications by authors named "Rune Hartmann"

73 Publications

SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity.

EBioMedicine 2021 Jun 4;68:103410. Epub 2021 Jun 4.

Department of Infectious Diseases, Aarhus University Hospital, Denmark; Department of Clinical Medicine, Aarhus University, Denmark.

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 3 and July 9 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).
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http://dx.doi.org/10.1016/j.ebiom.2021.103410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176920PMC
June 2021

Selective Janus kinase inhibition preserves interferon-λ-mediated antiviral responses.

Sci Immunol 2021 May;6(59)

Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany.

Inflammatory diseases are frequently treated with Janus kinase (JAK) inhibitors to diminish cytokine signaling. These treatments can lead to inadvertent immune suppression and may increase the risk of viral infection. Tyrosine kinase 2 (TYK2) is a JAK family member required for efficient type I interferon (IFN-α/β) signaling. We report here that selective TYK2 inhibition preferentially blocked potentially detrimental type I IFN signaling, whereas IFN-λ-mediated responses were largely preserved. In contrast, the clinically used JAK1/2 inhibitor baricitinib was equally potent in blocking IFN-α/β- or IFN-λ-driven responses. Mechanistically, we showed that epithelial cells did not require TYK2 for IFN-λ-mediated signaling or antiviral protection. TYK2 deficiency diminished IFN-α-induced protection against lethal influenza virus infection in mice but did not impair IFN-λ-mediated antiviral protection. Our findings suggest that selective TYK2 inhibitors used in place of broadly acting JAK1/2 inhibitors may represent a superior treatment option for type I interferonopathies to counteract inflammatory responses while preserving antiviral protection mediated by IFN-λ.
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http://dx.doi.org/10.1126/sciimmunol.abd5318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610871PMC
May 2021

Disparate temperature-dependent virus-host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium.

PLoS Biol 2021 03 29;19(3):e3001158. Epub 2021 Mar 29.

Institute of Virology and Immunology (IVI), Bern, Switzerland.

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.
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http://dx.doi.org/10.1371/journal.pbio.3001158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032198PMC
March 2021

Interferon lambda 4 genotype and pathway in alcoholic hepatitis.

Scand J Gastroenterol 2021 Mar 19;56(3):304-311. Epub 2021 Feb 19.

Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.

Objectives: Single nucleotide polymorphisms within the interferon lambda 4 ( gene influence liver inflammation and fibrosis in chronic liver disease. We investigated whether this is also the case during acute liver disease, alcoholic hepatitis. We, therefore, related variants within the gene to the clinical course of acute alcoholic hepatitis, and characterized the activation state of the IFN lambda system in these patients.

Methods: In this pilot study, 58 patients with alcoholic hepatitis were genotyped for the single nucleotide polymorphism (deltaG, deltaG/TT: IFN lambda 4 positive, TT/TT: IFN lambda 4 negative). The genotypes were related to mortality, infection and inflammation and expression of the IFNL receptor 1 and IFN inducible genes were measured in liver and peripheral leukocytes.

Results: Amongst the alcoholic hepatitis patients who died, the IFN negative patients live longer after diagnosis, and also the IFN negative patients tended to have an overall short-term survival benefit compared to IFN lambda positive patients ( = .058). The IFN lambda 4 negative patients at diagnosis had fewer circulating monocytes and lower plasma soluble CD163. The patients with alcoholic hepatitis had reduced expression of the IFNL receptor 1in both liver and blood compared with healthy controls. In blood, the expression of IFN stimulated genes was lower than in healthy controls and most so in the patients, who died.

Conclusions: The IFN lambda 4 pathway seems involved in the acute disease processes of alcoholic hepatitis and patients without IFN lambda expression seem to have a short-term survival benefit.
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http://dx.doi.org/10.1080/00365521.2021.1874046DOI Listing
March 2021

2'3'-cGAMP triggers a STING- and NF-κB-dependent broad antiviral response in .

Sci Signal 2020 12 1;13(660). Epub 2020 Dec 1.

Sino-French Hoffmann Institute, State Key Laboratory of Respiratory Disease, School of Basic Medical Science, Guangzhou Medical University, Guangzhou 511436, China.

We previously reported that an ortholog of STING regulates infection by picorna-like viruses in In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in and , genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.
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http://dx.doi.org/10.1126/scisignal.abc4537DOI Listing
December 2020

Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization.

Nucleic Acids Res 2020 11;48(20):11421-11433

Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark.

IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3. This positively charged surface also plays a crucial role in forming the active homodimer by interacting with the phosphorylated sites stabilizing the homodimer. Here, we describe a distinct molecular interaction that is responsible for adaptor docking and hence phosphorylation as well as a separate interaction responsible for the formation of active homodimer. We then demonstrate that IRF7 can be activated by both MAVS and STING in a manner highly similar to that of IRF3 but with one key difference. Regulation of IRF7 appears more tightly controlled; while a single phosphorylation event is sufficient to activate IRF3, at least two phosphorylation events are required for IRF7 activation.
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http://dx.doi.org/10.1093/nar/gkaa873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672473PMC
November 2020

SARS-CoV-2 evades immune detection in alveolar macrophages.

EMBO Rep 2020 12 28;21(12):e51252. Epub 2020 Oct 28.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

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.
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http://dx.doi.org/10.15252/embr.202051252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645910PMC
December 2020

STEEP mediates STING ER exit and activation of signaling.

Nat Immunol 2020 08 20;21(8):868-879. Epub 2020 Jul 20.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.
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http://dx.doi.org/10.1038/s41590-020-0730-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610351PMC
August 2020

Inhibition of SARS-CoV-2 by type I and type III interferons.

J Biol Chem 2020 10 25;295(41):13958-13964. Epub 2020 Jun 25.

Institute for Virology, FB10-Veterinary Medicine, Justus Liebig University, Giessen, Germany

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.
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http://dx.doi.org/10.1074/jbc.AC120.013788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549028PMC
October 2020

Systemic juvenile idiopathic arthritis and recurrent macrophage activation syndrome due to a CASP1 variant causing inflammasome hyperactivation.

Rheumatology (Oxford) 2020 10;59(10):3099-3105

Department of Infectious Diseases, Aarhus University Hospital.

Objectives: We investigated a patient with systemic juvenile idiopathic arthritis (sJIA) and recurrent macrophage activation syndrome (MAS) to discover genetic and immunological contributing factors.

Methods: Severe recurrent MAS motivated whole exome sequencing (WES) to identify genetic variants potentially involved in disease pathogenesis. In vitro peripheral blood mononuclear cell (PBMC) stimulations for cytokine expression and caspase-1 activity assays as well as NF-κB reporter luciferase assays were performed to functionally characterize variants.

Results: WES revealed an extremely rare heterozygous missense variant, c.482G>A, p.R161H in the CASP1 gene encoding pro-caspase-1. Lipopolysaccharide (LPS) stimulation of patient PBMCs induced high levels of IL-6 compared to controls, and activation of the NLRP3 inflammasome resulted in increased production of IL-1β and IL-18 as well as significantly elevated caspase-1 activity. Constitutive and inducible levels of IL-18 and IFNγ in whole blood were markedly elevated. Expression of the CASP1 variant in an NF-κB reporter luciferase assay induced increased NF-κB activation in a RIP2-dependent manner. The disease course of the patient was complicated by severe recurrent MAS. However, dual IL-1 and IL-6 blockade caused disease remission.

Conclusion: For the first time, we demonstrate the involvement of a CASP1 variant in sJIA and recurrent MAS. This variant is gain-of-function for both inflammasome and NF-κB activation leading to increased production of IL-6, IL-1β and IL-18. Although dual IL-1 and IL-6 blockade may be beneficial in patients, in whom single treatment is not sufficient to control MAS, caution should be practiced, since interstitial lung disease may progress despite apparent clinical and biochemical remission.
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http://dx.doi.org/10.1093/rheumatology/keaa242DOI Listing
October 2020

Type I and III interferons disrupt lung epithelial repair during recovery from viral infection.

Science 2020 08 11;369(6504):712-717. Epub 2020 Jun 11.

Immunoregulation Laboratory, The Francis Crick Institute, London, UK.

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-β) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-β 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).
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http://dx.doi.org/10.1126/science.abc2061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292500PMC
August 2020

COVID-19 and emerging viral infections: The case for interferon lambda.

J Exp Med 2020 05;217(5)

Division of Immunology, Division of Gastroenterology, Harvard Medical School, Boston Children's Hospital, Boston, MA.

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.
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http://dx.doi.org/10.1084/jem.20200653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155807PMC
May 2020

The Gene Is a Noncanonical Interferon Gene with a Unique but Evolutionarily Conserved Regulation.

J Virol 2020 02 14;94(5). Epub 2020 Feb 14.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Interferon lambda 4 (IFN-λ4) is a recently identified enigmatic member of the interferon (IFN) lambda family. Genetic data suggest that the gene acts in a proviral and anti-inflammatory manner in patients. However, the protein is indistinguishable from the other members of the interferon lambda family. We have investigated the gene regulation of in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the promoter. Thus, the regulation of the gene is radically different and might explain some of the atypical phenotypes associated with the gene in humans. Recent genetic evidence has highlighted how the gene acts in a counterintuitive manner, as patients with a nonfunctional gene exhibit increased clearance of hepatitis C virus (HCV) but also increased liver inflammation. This suggests that the gene acts in a proviral and anti-inflammatory manner. These surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the gene and its gene product, interferon lambda 4 (IFN-λ4). We have investigated the expression of the gene and found it to be poorly induced by viral infections. A thorough investigation of the promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of interferons (IFNs), i.e., the ability to be effectively induced by viral infections. We suggest that the unique function of the gene is related to its noncanonical transcriptional regulation.
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http://dx.doi.org/10.1128/JVI.01535-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022339PMC
February 2020

Length dependent activation of OAS proteins by dsRNA.

Cytokine 2020 02 17;126:154867. Epub 2019 Oct 17.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. Electronic address:

The oligoadenylate synthetase (OAS) family of enzymes are interferon-inducible antiviral proteins, which synthesize the secondary messenger 2'-5'-linked oligoadenosine (2-5A) in response to viral infection. The production of 2-5As induces RNA decay within the infected cells, thereby effectively preventing further viral replication. OAS shares structural similarity as well as the enzymatic mechanism with a different antiviral protein, cyclic GMP-AMP synthase (cGAS), but OAS is activated by dsRNA whereas cGAS is activated by dsDNA. Here, we have studied the structural requirement for the dsRNA activating OAS1 and OAS3, and compared it to recent studies on cGAS. We find that both OAS1 and OAS3, like cGAS, achieve their maximum activity with dsRNA molecules that are substantial longer than what one monomer of the enzyme can interact with. One molecule of OAS1 can cover approximately 18-20 base pairs of dsRNA, which is just short of two turns of a helix. However, RNAs of this length gave a very limited activity and the length dependency was even more pronounced for OAS3. Our data suggest that the OAS enzymes evolved to recognize long dsRNA as virally derived PAMPs, and that the length of the dsRNA is an important factor in discriminating self from non-self. Several structures of OAS1 bound to short dsRNAs exist, but our data show that OAS can only achieve minimal activity with these short activators (approximately 7-8% of maximal activity) and it is thus possible that these structures do not reveal the fully activated state of the OAS enzymes.
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http://dx.doi.org/10.1016/j.cyto.2019.154867DOI Listing
February 2020

Type I and Type III Interferons Differ in Their Adjuvant Activities for Influenza Vaccines.

J Virol 2019 12 13;93(23). Epub 2019 Nov 13.

Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany

Type I and type III interferons (IFNs) can promote adaptive immune responses in mice and improve vaccine-induced resistance to viral infections. The adjuvant effect of type III IFN (IFN-λ) specifically boosts mucosal immunity by an indirect mechanism, involving IFN-λ-induced production of thymic stromal lymphopoietin (TSLP), a cytokine that activates immune cells. To date, it remained unclear whether the previously described adjuvant effect of type I IFN (IFN-α/β) would also depend on TSLP and whether type I IFN stimulates different antibody subtypes. Here, we show that after infection with a live attenuated influenza virus, mice lacking functional type I IFN receptors failed to produce normal amounts of virus-specific IgG2c and IgA antibodies. In contrast, mice lacking functional IFN-λ receptors contained normal levels of virus-specific IgG2c but had reduced IgG1 and IgA antibody levels. When applied together with protein antigen, IFN-α stimulated the production of antigen-specific IgA and IgG2c to a greater extent than IgG1, irrespective of whether the mice expressed functional TSLP receptors and irrespective of whether the vaccine was applied by the intranasal or the intraperitoneal route. Taken together, these results demonstrate that the adjuvant activities of type I and type III IFNs are mechanistically distinct. Interferons can shape antiviral immune responses, but it is not well understood how they influence vaccine efficacy. We find that type I IFN preferentially promotes the production of antigen-specific IgG2c and IgA antibodies after infection with a live attenuated influenza virus or after immunization with influenza subunit vaccines. In contrast, type III IFN specifically enhances influenza virus-specific IgG1 and IgA production. The adjuvant effect of type I IFN was not dependent on TSLP, which is essential for the adjuvant effect of type III IFN. Type I IFN boosted vaccine-induced antibody production after immunization by the intranasal or the intraperitoneal route, whereas type III IFN exhibited its adjuvant activity only when the vaccine was delivered by the mucosal route. Our findings demonstrate that type I and type III IFNs trigger distinct pathways to enhance the efficacy of vaccines. This knowledge might be used to design more efficient vaccines against infectious diseases.
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http://dx.doi.org/10.1128/JVI.01262-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854507PMC
December 2019

What makes the hepatitis C virus evolve?

Elife 2019 09 3;8. Epub 2019 Sep 3.

Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, United States.

Polymorphisms in the gene that affect both the presence and the form of the coded protein are associated with changes in the hepatitis C virus.
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http://dx.doi.org/10.7554/eLife.50148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721787PMC
September 2019

Defective interferon priming and impaired antiviral responses in a patient with an IRF7 variant and severe influenza.

Med Microbiol Immunol 2019 Dec 6;208(6):869-876. Epub 2019 Jun 6.

Department of Infectious Diseases, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200, Aarhus N, Denmark.

Influenza infection is common worldwide with many individuals affected each year during epidemics and occasionally pandemics. Previous studies in animal models and a few human cases have established an important role of innate type I and III interferon (IFN) for viral elimination and mounting of antiviral responses. However, genetic and immunological determinants of very severe disseminated influenza virus infection in humans remain incompletely understood. Here, we describe an adult patient with severe influenza virus A (IAV) infection, in whom we identified a rare variant E331V in IFN regulatory factor (IRF)7 by whole-exome sequencing. Examination of patient cells demonstrated a cellular phenotype suggesting functional IRF7 impairment, since priming with IFN was almost abolished and IFN responses to IAV were significantly impaired in patient cells. Moreover, IAV replication was significantly higher in patient cells than in controls. Finally, expression of IRF7 E331V in HEK293 cells demonstrated significantly reduced activation of both IFNA7 and IFNB promoters in a luciferase reporter gene expression assay compared to IRF7 wild type. These findings provide further support for the essential role of IRF7 in amplifying antiviral IFN responses to ensure potent and sustained IFN responses during influenza virus infection in humans.
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http://dx.doi.org/10.1007/s00430-019-00623-8DOI Listing
December 2019

The Influence of the rs30461 Single Nucleotide Polymorphism on IFN-λ1 Activity and Secretion.

J Interferon Cytokine Res 2019 10 22;39(10):661-667. Epub 2019 May 22.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

Genetic variation within the loci is associated with several diseases and evidence indicates that the genes have been subjects of strong selection during recent human evolution. The nonsynonymous rs30461 single nucleotide polymorphism (SNP), generating interferon (IFN)-λ1 D188N, shows a strong signature of positive selection in European and Asian populations. Nevertheless, genetic association studies have failed to show any coupling of rs30461 to diseases such as psoriasis and periodontitis. Based on these observations, we purified IFN-λ1 N188 and IFN-λ1 D188 to compare the biological activity of these 2 IFN-λ1 versions. Furthermore, we evaluated the secretion of the 2 different IFN-λ1 versions. We were unable to observe any differences between IFN-λ1 N188 and IFN-λ1 D188 based on biological activity or secretion that could account for the positive selection.
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http://dx.doi.org/10.1089/jir.2019.0051DOI Listing
October 2019

Interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin.

Nat Immunol 2019 05 18;20(5):593-601. Epub 2019 Mar 18.

Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany.

Interferon-λ (IFN-λ) acts on mucosal epithelial cells and thereby confers direct antiviral protection. In contrast, the role of IFN-λ in adaptive immunity is far less clear. Here, we report that mice deficient in IFN-λ signaling exhibited impaired CD8 T cell and antibody responses after infection with a live-attenuated influenza virus. Virus-induced release of IFN-λ triggered the synthesis of thymic stromal lymphopoietin (TSLP) by M cells in the upper airways that, in turn, stimulated migratory dendritic cells and boosted antigen-dependent germinal center reactions in draining lymph nodes. The IFN-λ-TSLP axis also boosted production of the immunoglobulins IgG1 and IgA after intranasal immunization with influenza virus subunit vaccines and improved survival of mice after challenge with virulent influenza viruses. IFN-λ did not influence the efficacy of vaccines applied by subcutaneous or intraperitoneal routes, indicating that IFN-λ plays a vital role in potentiating adaptive immune responses that initiate at mucosal surfaces.
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http://dx.doi.org/10.1038/s41590-019-0345-xDOI Listing
May 2019

Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ.

J Interferon Cytokine Res 2018 11 18;38(11):469-479. Epub 2018 Oct 18.

1 de Duve Institute , Université Catholique de Louvain, Brussels, Belgium .

The type III interferon (IFN-λ) family includes 4 IFN-λ subtypes in man. In the mouse, only the genes coding for IFN-λ2 and -λ3 are present. Unlike mouse and human type I IFNs (IFN-α/β), which exhibit strong species specificity, type III IFNs were reported to act in a cross-specific manner. We reexamined the cross-specificity and observed that mouse and human IFN-λ exhibit some species specificity, although much less than type I IFNs. Mouse IFN-λ3 displayed clear species specificity, being 25-fold less active in human cells than the closely related mouse IFN-λ2. This specificity likely depends on amino acids in α helices A and F that diverged from other IFN-λ sequences. Human IFN-λ4, in contrast, retained high activity in mouse cells. We next developed a firefly luciferase-based reporter cell line, named Fawa-λ-luc, to detect IFN-λ in biological fluids with high specificity and sensitivity. Fawa-λ-luc cells, derived from mouse epithelial cells that are responsive to IFN-λ, were made nonresponsive to type I IFNs by inactivation of the Ifnar2 gene and strongly responsive to IFN-λ by overexpression of the mouse IFNLR1. This bioassay was as sensitive as a commercially available enzyme-linked immunosorbent assay in detecting mouse IFN-λ in cell culture supernatant, as well as in serum and bronchoalveolar lavage samples of virus-infected mice. The assay also enabled the sensitive detection of human IFN-λ activity, including that of the divergent IFN-λ4 with a bias, however, due to variable activity of IFN-λ subtypes.
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http://dx.doi.org/10.1089/jir.2018.0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249671PMC
November 2018

The role of IFN in the development of NAFLD and NASH.

Cytokine 2019 12 21;124:154519. Epub 2018 Aug 21.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. Electronic address:

Non-alcoholic fatty liver disease (NAFLD) and its progressive inflammatory form non-alcoholic steatohepatitis (NASH) are major health challenges due to a significant increase in their incidence and prevalence. While NAFLD is largely benign, the chronic liver inflammation in NASH patients may cause progression to liver cirrhosis and hepatocellular carcinoma. There is an urgent need for a better understanding of the factors, which drive the progression from NAFLD to NASH and how to use this information both to improve diagnostic and to develop new treatment strategies. Increasing evidence points to interferons (IFNs) as key players in NAFLD and particular in the progression to NASH. IFNs crucial role in disease development is supported by both genetic evidence and animal studies. In this review, we describe the involvement of both type I and type III IFNs in the development and progression of NAFLD and NASH.
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http://dx.doi.org/10.1016/j.cyto.2018.08.013DOI Listing
December 2019

The Kinase IKKβ Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Immunity 2018 08 14;49(2):225-234.e4. Epub 2018 Aug 14.

Université de Strasbourg, CNRS, Insect Models of Innate Immunity (M3I; UPR9022), 67084 Strasbourg, France; Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Guangzhou 511436, China. Electronic address:

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKβ and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.
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http://dx.doi.org/10.1016/j.immuni.2018.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267954PMC
August 2018

IFN-λ prevents influenza virus spread from the upper airways to the lungs and limits virus transmission.

Elife 2018 04 13;7. Epub 2018 Apr 13.

Institute of Virology, Medical Center, University of Freiburg, Freiburg, Germany.

Host factors restricting the transmission of respiratory viruses are poorly characterized. We analyzed the contribution of type I and type III interferon (IFN) using a mouse model in which the virus is selectively administered to the upper airways, mimicking a natural respiratory virus infection. Mice lacking functional IFN-λ receptors () no longer restricted virus dissemination from the upper airways to the lungs. mice shed significantly more infectious virus particles via the nostrils and transmitted the virus much more efficiently to naïve contacts compared with wild-type mice or mice lacking functional type I IFN receptors. Prophylactic treatment with IFN-α or IFN-λ inhibited initial virus replication in all parts of the respiratory tract, but only IFN-λ conferred long-lasting antiviral protection in the upper airways and blocked virus transmission. Thus, IFN-λ has a decisive and non-redundant function in the upper airways that greatly limits transmission of respiratory viruses to naïve contacts.
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http://dx.doi.org/10.7554/eLife.33354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953542PMC
April 2018

The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi's sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein.

PLoS Pathog 2018 03 2;14(3):e1006937. Epub 2018 Mar 2.

Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.
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http://dx.doi.org/10.1371/journal.ppat.1006937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851652PMC
March 2018

Frequently used bioinformatics tools overestimate the damaging effect of allelic variants.

Genes Immun 2019 01 4;20(1):10-22. Epub 2017 Dec 4.

Department of Molecular Biology and Genetics, Aarhus University, 8000, Aarhus, Denmark.

We selected two sets of naturally occurring human missense allelic variants within innate immune genes. The first set represented eleven non-synonymous variants in six different genes involved in interferon (IFN) induction, present in a cohort of patients suffering from herpes simplex encephalitis (HSE) and the second set represented sixteen allelic variants of the IFNLR1 gene. We recreated the variants in vitro and tested their effect on protein function in a HEK293T cell based assay. We then used an array of 14 available bioinformatics tools to predict the effect of these variants upon protein function. To our surprise two of the most commonly used tools, CADD and SIFT, produced a high rate of false positives, whereas SNPs&GO exhibited the lowest rate of false positives in our test. As the problem in our test in general was false positive variants, inclusion of mutation significance cutoff (MSC) did not improve accuracy.
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http://dx.doi.org/10.1038/s41435-017-0002-zDOI Listing
January 2019

cGAS is activated by DNA in a length-dependent manner.

EMBO Rep 2017 10 10;18(10):1707-1715. Epub 2017 Aug 10.

Department of Biomedicine, Aarhus University, Aarhus, Denmark

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.
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http://dx.doi.org/10.15252/embr.201744017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623850PMC
October 2017