Publications by authors named "Peter Staeheli"

143 Publications

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

Different effects of constitutive and induced microbiota modulation on microglia in a mouse model of Alzheimer's disease.

Acta Neuropathol Commun 2020 07 29;8(1):119. Epub 2020 Jul 29.

Institute of Neuropathology, University of Freiburg, Breisacher Str. 64, 79106, Freiburg, Germany.

It was recently revealed that gut microbiota promote amyloid-beta (Aβ) burden in mouse models of Alzheimer's disease (AD). However, the underlying mechanisms when using either germ-free (GF) housing conditions or treatments with antibiotics (ABX) remained unknown. In this study, we show that GF and ABX-treated 5x familial AD (5xFAD) mice developed attenuated hippocampal Aβ pathology and associated neuronal loss, and thereby delayed disease-related memory deficits. While Aβ production remained unaffected in both GF and ABX-treated 5xFAD mice, we noticed in GF 5xFAD mice enhanced microglial Aβ uptake at early stages of the disease compared to ABX-treated 5xFAD mice. Furthermore, RNA-sequencing of hippocampal microglia from SPF, GF and ABX-treated 5xFAD mice revealed distinct microbiota-dependent gene expression profiles associated with phagocytosis and altered microglial activation states. Taken together, we observed that constitutive or induced microbiota modulation in 5xFAD mice differentially controls microglial Aβ clearance mechanisms preventing neurodegeneration and cognitive deficits.
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http://dx.doi.org/10.1186/s40478-020-00988-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389451PMC
July 2020

Antagonism of interferon signaling by fibroblast growth factors promotes viral replication.

EMBO Mol Med 2020 09 27;12(9):e11793. Epub 2020 Jul 27.

Department of Biology, Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.

Fibroblast growth factors (FGFs) play key roles in the pathogenesis of different human diseases, but the cross-talk between FGFs and other cytokines remains largely unexplored. We identified an unexpected antagonistic effect of FGFs on the interferon (IFN) signaling pathway. Genetic or pharmacological inhibition of FGF receptor signaling in keratinocytes promoted the expression of interferon-stimulated genes (ISG) and proteins in vitro and in vivo. Conversely, FGF7 or FGF10 treatment of keratinocytes suppressed ISG expression under homeostatic conditions and in response to IFN or poly(I:C) treatment. FGF-mediated ISG suppression was independent of IFN receptors, occurred at the transcriptional level, and required FGF receptor kinase and proteasomal activity. It is not restricted to keratinocytes and functionally relevant, since FGFs promoted the replication of herpes simplex virus I (HSV-1), lymphocytic choriomeningitis virus, and Zika virus. Most importantly, inhibition of FGFR signaling blocked HSV-1 replication in cultured human keratinocytes and in mice. These results suggest the use of FGFR kinase inhibitors for the treatment of viral infections.
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http://dx.doi.org/10.15252/emmm.201911793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507082PMC
September 2020

Interferon-λ Receptor Expression: Novel Reporter Mouse Reveals Within- and Cross-Tissue Heterogeneity.

J Interferon Cytokine Res 2020 06 4;40(6):292-300. Epub 2020 May 4.

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

Interferon-λ (IFN-λ) plays an important role in mucosal immunity, but reliable information regarding the expression of the IFN-λ receptor in individual cells is still missing. One reason for this knowledge gap is the lack of antibodies that specifically recognize the unique IFNLR1 subunit of the dimeric IFN-λ receptor complex. In this study, we investigated whether a reporter mouse carrying a bacterial β-galactosidase gene inserted into the locus could be used to visualize IFN-λ receptor-expressing cells in whole organs. First we confirmed that insertion of the reporter cassette inactivated the gene, and that gene function could be restored by removing the β-galactosidase insert by site-specific recombination. When whole tissues were analyzed, prominent β-galactosidase activity was confined to the intestinal tract of reporter mice. However, only the snout expressed β-galactosidase at levels high enough for reliable detection in whole tissue extracts. Interestingly, individual epithelial cells in the upper airways expressed β-galactosidase activity to variable degrees as determined by flow cytometry and histology, suggesting a remarkable heterogeneity in IFNLR1 expression levels. Taken together, our results demonstrate a surprisingly strong within- and cross-tissue heterogeneity of IFNLR1 expression that may have physiological implications.
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http://dx.doi.org/10.1089/jir.2019.0265DOI Listing
June 2020

Prevention of influenza virus infection and transmission by intranasal administration of a porous maltodextrin nanoparticle-formulated vaccine.

Int J Pharm 2020 May 20;582:119348. Epub 2020 Apr 20.

Inserm, LIRIC - UMR 995, F-59 000 Lille, France; University of Lille, LIRIC - UMR 995, F-59 000 Lille, France; CHRU of Lille, LIRIC - UMR 995, F-59 000 Lille, France; University of Artois, 62300 Lens, France.

Influenza vaccines administered intramuscularly exhibit poor mucosal immune responses in the respiratory tract which is the prime site of the infection. Intranasal vaccination is a potential route for vaccine delivery which has been demonstrated effective in inducing protective immune responses in both systemic and mucosal compartments. For this purpose, nanoparticles have been used as antigen delivery systems to improve antigen capture by immune cells. In this paper we demonstrate efficient delivery of viral antigens to airway epithelial cells, macrophages and dendritic cells, using polysaccharide nanoparticles (NPL), leading to a strong protection against influenza virus infection. A formulation combining split Udorn virus antigens with NPL and the mucosal protein adjuvant CTA1-DD was administered intranasally and resulted in an enhanced specific humoral immune response. Furthermore, NPL carrying split Udorn, with or without CTA1-DD, inhibited virus transmission from infected to uninfected naive mice. These results demonstrate that an intranasal delivery system combining NPL, mucosal adjuvant CTA1-DD and split virus antigens confers robust protection against influenza infection and inhibits virus transmission.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119348DOI Listing
May 2020

A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control.

J Hepatol 2020 05 15;72(5):960-975. Epub 2020 Jan 15.

Institute of Experimental Immunology, Bonn, Germany.

Background & Aims: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver-resident macrophages. However, hepatocytes, the parenchymal cells of the liver, also possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct antiviral mechanisms employed by hepatocytes.

Methods: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (Ikkβ) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/β signaling-(Ifnar), or interferon-α/β signaling in myeloid cells-(Ifnar) were infected.

Results: Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1 signaling but rather on Toll-like receptor signaling. LCMV-infected Ikkβ livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8 T cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKβ, demonstrating a hepatocyte-intrinsic effect. Similar to livers of Ikkβ mice, enhanced hepatocytic LCMV accumulation was observed in livers of Ifnar mice, whereas Ifnar mice were able to control LCMV infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/β-mediated inhibition of HBV replication in vitro.

Conclusions: Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/β signaling, which is pivotal for strong early ISG responses, immune cell infiltration and hepatic viral clearance.

Lay Summary: Innate immune cells have been ascribed a primary role in controlling viral clearance upon hepatic infections. We identified a novel dual role for NF-κB signaling in infected hepatocytes which was crucial for maximizing interferon responses and initiating adaptive immunity, thereby efficiently controlling hepatic virus replication.
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http://dx.doi.org/10.1016/j.jhep.2019.12.019DOI Listing
May 2020

The alternative cap-binding complex is required for antiviral defense in vivo.

PLoS Pathog 2019 12 19;15(12):e1008155. Epub 2019 Dec 19.

Institute of Virology, Technical University of Munich, School of Medicine, Munich, Germany.

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.
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http://dx.doi.org/10.1371/journal.ppat.1008155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946169PMC
December 2019

Influenza restriction factor MxA functions as inflammasome sensor in the respiratory epithelium.

Sci Immunol 2019 10;4(40)

Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

The respiratory epithelium is exposed to the environment and initiates inflammatory responses to exclude pathogens. Influenza A virus (IAV) infection triggers inflammatory responses in the respiratory mucosa, but the mechanisms of inflammasome activation are poorly understood. We identified MxA as a functional inflammasome sensor in respiratory epithelial cells that recognizes IAV nucleoprotein and triggers the formation of ASC (apoptosis-associated speck-like protein containing a CARD) specks via interaction of its GTPase domain with the PYD domain of ASC. ASC specks were present in bronchiolar epithelial cells of IAV-infected MxA-transgenic mice, which correlated with early IL-1β production and early recruitment of granulocytes in the lungs of infected mice. Collectively, these results demonstrate that MxA contributes to IAV resistance by triggering a rapid inflammatory response in infected respiratory epithelial cells.
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http://dx.doi.org/10.1126/sciimmunol.aau4643DOI Listing
October 2019

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

Microbiota-Driven Tonic Interferon Signals in Lung Stromal Cells Protect from Influenza Virus Infection.

Cell Rep 2019 07;28(1):245-256.e4

Immunoregulation Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

Type I interferon (IFNα/β) pathways are fine-tuned to elicit antiviral protection while minimizing immunopathology; however, the initiating stimuli, target tissues, and underlying mechanisms are unclear. Using models of physiological and dysregulated IFNα/β receptor (IFNAR1) surface expression, we show here that IFNAR1-dependent signals set the steady-state IFN signature in both hematopoietic and stromal cells. Increased IFNAR1 levels promote a lung environment refractory to early influenza virus replication by elevating the baseline interferon signature. Commensal microbiota drive the IFN signature specifically in lung stroma, as shown by antibiotic treatment and fecal transplantation. Bone marrow chimera experiments identify lung stromal cells as crucially important for early antiviral immunity and stroma-immune cell interaction for late antiviral resistance. We propose that the microbiota-driven interferon signature in lung epithelia impedes early virus replication and that IFNAR1 surface levels fine-tune this signature. Our findings highlight the interplay between bacterial and viral exposure, with important implications for antibiotic use.
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http://dx.doi.org/10.1016/j.celrep.2019.05.105DOI Listing
July 2019

Interferon-λ orchestrates innate and adaptive mucosal immune responses.

Nat Rev Immunol 2019 10 14;19(10):614-625. Epub 2019 Jun 14.

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

Type III interferon (IFN-λ) was initially thought to have functions similar to those of the type I interferons (IFN-α and IFN-β). New findings have indicated, however, that IFN-λ has a non-redundant role in the innate antiviral, antifungal and antiprotozoal defences of mucosal barriers. In this Review, we highlight recent work showing that IFN-λ inhibits virus dissemination within the body and limits the transmission of respiratory and gastrointestinal viruses to naive hosts. We also discuss findings indicating that IFN-λ can act on neutrophils to prevent invasive pulmonary aspergillosis. We summarize results showing that IFN-λ signalling differs in several respects from IFN-α and IFN-β signalling, particularly in neutrophils. Finally, we discuss new findings indicating that IFN-λ is a potent enhancer of adaptive immune responses in the respiratory mucosa.
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http://dx.doi.org/10.1038/s41577-019-0182-zDOI 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

Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections.

Front Immunol 2018 12;9:2060. Epub 2018 Sep 12.

Mucosal Immunobiology and Vaccine Center, Department of Microbiology and Immunology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Due to the high risk of an outbreak of pandemic influenza, the development of a broadly protective universal influenza vaccine is highly warranted. The design of such a vaccine has attracted attention and much focus has been given to nanoparticle-based influenza vaccines which can be administered intranasally. This is particularly interesting since, contrary to injectable vaccines, mucosal vaccines elicit local IgA and lung resident T cell immunity, which have been found to correlate with stronger protection in experimental models of influenza virus infections. Also, studies in human volunteers have indicated that pre-existing CD4 T cells correlate well to increased resistance against infection. We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. To improve the protective ability of the self-adjuvanting fusion protein, CTA1-3M2e-DD, we incorporated it into porous maltodextrin nanoparticles (NPLs). This proof-of-principle study demonstrates that the combined vaccine vector given intranasally enhanced immune protection against a live challenge infection and reduced the risk of virus transmission between immunized and unimmunized individuals. Most importantly, immune responses to NPLs that also contained recombinant hemagglutinin (HA) were strongly enhanced in a CTA1-enzyme dependent manner and we achieved broadly protective immunity against a lethal infection with heterosubtypic influenza virus. Immune protection was mediated by enhanced levels of lung resident CD4 T cells as well as anti-HA and -M2e serum IgG and local IgA antibodies.
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http://dx.doi.org/10.3389/fimmu.2018.02060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146233PMC
September 2019

Human MX2/MxB: a Potent Interferon-Induced Postentry Inhibitor of Herpesviruses and HIV-1.

J Virol 2018 12 27;92(24). Epub 2018 Nov 27.

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

Interferons limit viral replication by inducing intracellular restriction factors, such as the GTPase MxB (also designated MX2), which inhibits HIV-1 and, as recently shown, herpesviruses. Inhibition of these viruses occurs at ill-defined steps after viral entry and requires formation of MxB dimers or oligomers, but GTP hydrolysis is needed only for blocking herpesviruses. Together with previous findings on related MxA, the new research on MxB highlights the mechanistic diversity by which MX proteins interfere with viral replication.
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http://dx.doi.org/10.1128/JVI.00709-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258936PMC
December 2018

Passively transferred M2e-specific monoclonal antibody reduces influenza A virus transmission in mice.

Antiviral Res 2018 10 1;158:244-254. Epub 2018 Sep 1.

VIB Center for Medical Biotechnology, VIB, Technologiepark 927, Ghent, B-9052, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, B-9052, Belgium. Electronic address:

Influenza represents a global public health threat. Currently available influenza vaccines are effective against strain-matched influenza A and B viruses but do not protect against novel pandemic viruses. Vaccine candidates that target conserved B or T cell epitopes of influenza viruses could circumvent this shortcoming. The conserved extracellular domain of matrix protein 2 (M2e) of influenza A is an example of such a broadly protective vaccine candidate. Protection by M2e-based vaccine candidates largely depends on M2e-specific IgG antibodies. Here we show that the M2e-specific IgG2a monoclonal antibody 65 (MAb 65) can reduce influenza A/Udorn/72 (H3N2) and A/Hong Kong/68 (H3N2) virus plaque formation. This effect was not observed with other influenza A virus strains tested. We further show that passive transfer of MAb 65 to mice can reduce viral loads in the upper and lower airways, which results in reduced transmission of A/Udorn/72 and A/Hong Kong/68 viruses to cohoused, unimmunized contact mice. Virus restriction by passively transferred Mab 65 was significantly less pronounced in Fcgr1Fcgr3 mutant mice compared with wild type controls, suggesting that in vivo protection provided by MAb 65 depends on Fcγ receptor-mediated antibody effector mechanisms. We conclude that M2e-based antibody immune therapy has the potential to diminish influenza A virus replication in the immunized host as well as in exposed naïve contacts.
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http://dx.doi.org/10.1016/j.antiviral.2018.08.017DOI Listing
October 2018

Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice.

PLoS Pathog 2018 08 3;14(8):e1007235. Epub 2018 Aug 3.

Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research, Braunschweig, and the Hannover Medical School, Hannover, Germany.

During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-β reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-β responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR-/- mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.
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http://dx.doi.org/10.1371/journal.ppat.1007235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107283PMC
August 2018

The Discovery of the Antiviral Resistance Gene Mx: A Story of Great Ideas, Great Failures, and Some Success.

Annu Rev Virol 2018 09 29;5(1):33-51. Epub 2018 Jun 29.

Institute of Virology, Medical Center University of Freiburg, D-79104 Freiburg, Germany; email:

The discovery of the Mx gene-dependent, innate resistance of mice against influenza virus was a matter of pure chance. Although the subsequent analysis of this antiviral resistance was guided by straightforward logic, it nevertheless led us into many blind alleys and was full of surprising turns and twists. Unexpectedly, this research resulted in the identification of one of the first interferon-stimulated genes and provided a new view of interferon action. It also showed that in many species, MX proteins have activities against a broad range of viruses. To this day, Mx research continues to flourish and to provide insights into the never-ending battle between viruses and their hosts.
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http://dx.doi.org/10.1146/annurev-virology-092917-043525DOI Listing
September 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

Oxeiptosis, a ROS-induced caspase-independent apoptosis-like cell-death pathway.

Nat Immunol 2018 02 18;19(2):130-140. Epub 2017 Dec 18.

Innate Immunity Laboratory, Max-Planck Institute of Biochemistry, Martinsried, Munich, Germany.

Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5 mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5 mice. This pathway, which we have called 'oxeiptosis', was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.
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http://dx.doi.org/10.1038/s41590-017-0013-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786482PMC
February 2018

License to kill: IFN-λ regulates antifungal activity of neutrophils.

Sci Immunol 2017 11 17;2(17). Epub 2017 Nov 17.

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

Interferon-λ mediates antifungal immunity by stimulating neutrophils to generate reactive oxygen species.
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http://dx.doi.org/10.1126/sciimmunol.aap9614DOI Listing
November 2017

RIG-I Activation Protects and Rescues from Lethal Influenza Virus Infection and Bacterial Superinfection.

Mol Ther 2017 09 8;25(9):2093-2103. Epub 2017 Jul 8.

Department of Otolaryngology, University Hospital Bonn, 53127 Bonn, Germany.

Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5'-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7 days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589155PMC
September 2017

In vivo evasion of MxA by avian influenza viruses requires human signature in the viral nucleoprotein.

J Exp Med 2017 05 10;214(5):1239-1248. Epub 2017 Apr 10.

Institute of Virology, Medical Center University of Freiburg, 79106 Freiburg, Germany

Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous genes but instead carries the human locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.
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http://dx.doi.org/10.1084/jem.20161033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413327PMC
May 2017

Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma.

Nat Commun 2017 02 15;8. Epub 2017 Feb 15.

Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1.

Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.
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http://dx.doi.org/10.1038/ncomms14278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330852PMC
February 2017

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

J Virol 2017 04 13;91(7). Epub 2017 Mar 13.

Medical Biotechnology Center, VIB, Ghent, Belgium

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors , whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, , , and BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies. There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different protective potential that is controlled by their capacity to interact with activating Fcγ receptors.
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http://dx.doi.org/10.1128/JVI.02500-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355615PMC
April 2017

Viral vector vaccines protect cockatiels from inflammatory lesions after heterologous parrot bornavirus 2 challenge infection.

Vaccine 2017 01 22;35(4):557-563. Epub 2016 Dec 22.

Institute for Virology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany. Electronic address:

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), a chronic neurologic and often fatal disorder of psittacines including endangered species. To date no causative therapy or immunoprophylaxis is available. Our previous work has shown that viral vector vaccines can delay the course of homologous bornavirus challenge infections but failed to protect against PDD when persistent infection was not prevented. The goal of this study was to refine our avian bornavirus vaccination and infection model to better represent natural bornavirus infections in order to achieve full protection against a heterologous challenge infection. We observed that parrot bornavirus 2 (PaBV-2) readily infected cockatiels (Nymphicus hollandicus) by combined intramuscular and subcutaneous injection with as little as 10foci-forming units (ffu) per bird, whereas a 500-fold higher dose of the same virus administered via peroral and oculonasal route did not result in persistent infection. These results indicated that experimental bornavirus challenge infections with this virus should be performed via the parenteral route. Prime-boost vaccination of cockatiels with Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) vectors expressing the nucleoprotein and phosphoprotein genes of PaBV-4 substantially blocked bornavirus replication following parenteral challenge infection with 10ffu of heterologous PaBV-2. Only two out of six vaccinated birds had very low viral levels detectable in a few organs. As a consequence, only one vaccinated bird developed mild PDD-associated microscopic lesions, while mock-vaccinated controls were not protected against PaBV-2 infection and inflammation. Our results demonstrate that NDV and MVA vector vaccines can protect against invasive heterologous bornavirus challenge infections and subsequent PDD. These vector vaccines represent a promising tool to combat avian bornaviruses in psittacine populations.
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http://dx.doi.org/10.1016/j.vaccine.2016.12.022DOI Listing
January 2017

Epithelial Barriers in Murine Skin during Herpes Simplex Virus 1 Infection: The Role of Tight Junction Formation.

J Invest Dermatol 2017 04 6;137(4):884-893. Epub 2016 Dec 6.

Center for Biochemistry, University of Cologne, Cologne, Germany; Department of Dermatology, University of Cologne, Cologne, Germany. Electronic address:

Herpes simplex virus 1 has to overcome skin or mucosa barriers to infect its human host. The impact of the various barrier functions on successful viral invasion is not known. On ex vivo infection of murine skin, we observed efficient invasion only via the basal epidermal layer when the dermis was removed. Here, we investigated how wounding and intercellular junction formation control successful viral entry. After wounding of skin samples or removal of the stratum corneum, infected cells were rarely detected. On the basis of infection studies in epidermis from IFN-stimulated mice, we assume that mechanical wounding does not lead to an antiviral state that impedes infection. When we infected human skin equivalents, we observed entry only into unstratified keratinocytes or after wounding of fully stratified cultures. Reduced infection of keratinocytes after calcium-induced stratification confirmed the impact of junction formation. To assess the effect of functional tight junctions, stratified cultures of polarity regulator partitioning-defective-3- or E-cadherin-deficient keratinocytes were infected. As the number of infected cells strongly increased with enhanced paracellular permeability, we conclude that the formation of functional tight junctions interferes with viral entry indicating that next to the stratum corneum tight junctions are a major physical barrier for herpes simplex virus 1 invasion into tissue.
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http://dx.doi.org/10.1016/j.jid.2016.11.027DOI Listing
April 2017

Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries.

Sci Rep 2016 11 10;6:36840. Epub 2016 Nov 10.

Institute for Virology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.
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http://dx.doi.org/10.1038/srep36840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103271PMC
November 2016

Influenza Virus Susceptibility of Wild-Derived CAST/EiJ Mice Results from Two Amino Acid Changes in the MX1 Restriction Factor.

J Virol 2016 Dec 14;90(23):10682-10692. Epub 2016 Nov 14.

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

The interferon-regulated Mx1 gene of the A2G mouse strain confers a high degree of resistance against influenza A and Thogoto viruses. Most other laboratory inbred mouse strains carry truncated nonfunctional Mx1 alleles and, consequently, exhibit high virus susceptibility. Interestingly, CAST/EiJ mice, derived from wild Mus musculus castaneus, possess a seemingly intact Mx1 gene but are highly susceptible to influenza A virus challenge. To determine whether the enhanced influenza virus susceptibility is due to intrinsically reduced antiviral activity of the CAST-derived Mx1 allele, we generated a congenic C57BL/6J mouse line that carries the Mx locus of CAST/EiJ mice. Adult animals of this line were almost as susceptible to influenza virus challenge as standard C57BL/6J mice lacking functional Mx1 alleles but exhibited far more pronounced resistance to Thogoto virus. Sequencing revealed that CAST-derived MX1 differs from A2G-derived MX1 by two amino acids (G83R and A222V) in the GTPase domain. Especially the A222V mutation reduced GTPase activity of purified MX1 and diminished the inhibitory effect of MX1 in influenza A virus polymerase activity assays. Further, MX1 protein was substantially less abundant in organs of interferon-treated mice carrying the CAST Mx1 allele than in those of mice carrying the A2G Mx1 allele. We found that the CAST-specific mutations reduced the metabolic stability of the MX1 protein although Mx1 mRNA levels were unchanged. Thus, the enhanced influenza virus susceptibility of CAST/EiJ mice can be explained by minor alterations in the MX1 restriction factor that negatively affect its enzymatic activity and reduce its half-life.

Importance: Although the crystal structure of the prototypic human MXA protein is known, the importance of specific protein domains for antiviral activity is still incompletely understood. Novel insights might come from studying naturally occurring MX protein variants with altered antiviral activity. Here we identified two seemingly minor amino acid changes in the GTPase domain that negatively affect the enzymatic activity and metabolic stability of murine MX1 and thus dramatically reduce the influenza virus resistance of the respective mouse inbred strain. These observations highlight our current inability to predict the biological consequences of previously uncharacterized MX mutations in mice. Since this is probably also true for naturally occurring mutations in Mx genes of humans, careful experimental analysis of any natural MXA variants for altered activity is necessary in order to assess possible consequences of such mutations on innate antiviral immunity.
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http://dx.doi.org/10.1128/JVI.01213-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110186PMC
December 2016

Synergistic antiviral activity of ribavirin and interferon-α against parrot bornaviruses in avian cells.

J Gen Virol 2016 09 20;97(9):2096-2103. Epub 2016 Jul 20.

Institute for Virology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany.

Avian bornaviruses are the causative agents of proventricular dilatation disease (PDD), a widely distributed and often fatal disease in captive psittacines. Because neither specific prevention measures nor therapies against PDD and bornavirus infections are currently available, new antiviral strategies are required to improve animal health. We show here that the nucleoside analogue ribavirin inhibited bornavirus activity in a polymerase reconstitution assay and reduced viral load in avian cell lines infected with two different parrot bornaviruses. Furthermore, we observed that ribavirin enhanced type I IFN signalling in avian cells. Combined treatment of avian bornavirus-infected cells with ribavirin and recombinant IFN-α strongly enhanced the antiviral efficiency compared to either drug alone. The combined use of ribavirin and type I IFN might represent a promising new strategy for therapeutic treatment of captive parrots persistently infected with avian bornaviruses.
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http://dx.doi.org/10.1099/jgv.0.000555DOI Listing
September 2016