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Interferons are essential for innate and adaptive immune responses against a wide variety of pathogens. Interferon lambda (IFN-λ) protects mucosal barriers during pathogen exposure. The intestinal epithelium is the first contact site forToxoplasma gondii(T. gondii) with its hosts and the first defense line that limits parasite infection. Knowledge of very earlyT. gondiiinfection events in the gut tissue is limited and a possible contribution of IFN-λ has not been investigated so far. Here, we demonstrate with systemic interferon lambda receptor (IFNLR1) and conditional (Villin-Cre) knockout mouse models and bone marrow chimeras of oralT. gondiiinfection and mouse intestinal organoids a significant impact of IFN-λ signaling in intestinal epithelial cells and neutrophils toT. gondiicontrol in the gastrointestinal tract. Our results expand the repertoire of interferons that contribute to the control ofT. gondiiand may lead to novel therapeutic approaches against this world-wide zoonotic pathogen.

Poultry passport to pandemic What conditions are required to nurture the seeds of a pandemic? The avian influenza virus H7N9 rarely spills over into humans, but when it does, mortality exceeds 30%, far in excess of that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Chen et al . used whole-genome sequencing to investigate the contribution of rare mutations among poultry workers who can be exposed to high levels of H7N9. Multiple defective single-nucleotide variants in the myxovirus resistance Mx1 locus were prevalent in H7N9 patients. In vitro infection experiments and influenza polymerase activity assays showed that 14 of the 17 MxA protein variants had no antiviral activity. Thus, individuals with such genetic vulnerabilities, when exposed to high virus loads, may act as crucibles for transmission of virulent new influenza subtypes. —CA

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 Ifnlr1 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 Ifnlr1 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.

Understanding the physiological origins of age-related cognitive decline is of critical importance given the rising age of the world’s population. Previous work in animal models has established a strong link between cognitive performance and the microbiota, and it is known that the microbiome undergoes profound remodeling in older adults. Despite growing evidence for the association between age-related cognitive decline and changes in the gut microbiome, the mechanisms underlying such interactions between the brain and the gut are poorly understood. Here, using fecal microbiota transplantation (FMT), we demonstrate that age-related remodeling of the gut microbiota leads to decline in cognitive function in mice and that this impairment can be rescued by transplantation of microbiota from young animals. Moreover, using a metabolomic approach, we found elevated concentrations of δ-valerobetaine, a gut microbiota-derived metabolite, in the blood and brain of aged mice and older adults. We then demonstrated that δ-valerobetaine is deleterious to learning and memory processes in mice. At the neuronal level, we showed that δ-valerobetaine modulates inhibitory synaptic transmission and neuronal network activity. Finally, we identified specific bacterial taxa that significantly correlate with δ-valerobetaine levels in the brain. Based on our findings, we propose that δ-valerobetaine contributes to microbiota-driven brain aging and that the associated mechanisms represent a promising target for countering age-related cognitive decline.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2–neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti–COVID-19 biologic that is now being evaluated in the clinic. ispartof: SCIENCE TRANSLATIONAL MEDICINE vol:13 issue:621 ispartof: location:United States status: published

A pivotal role of type I interferons in systemic lupus erythematosus (SLE) is widely accepted. Type III interferons (IFN-λ) however, the most recently discovered cytokines grouped within the interferon family, have not been extensively studied in lupus disease models yet. Growing evidence suggests a role for IFN-λ in regulating both innate and adaptive immune responses, and increased serum concentrations have been described in multiple autoimmune diseases including SLE. Using the pristane-induced lupus model, we found that mice with defective IFN-λ receptors (Ifnlr1−/−) showed increased survival rates, decreased lipogranuloma formation and reduced anti-dsDNA autoantibody titers in the early phase of autoimmunity development compared to pristane-treated wild-type mice. Moreover, Ifnlr1−/− mice treated with pristane had reduced numbers of inflammatory mononuclear phagocytes and cNK cells in their kidneys, resembling untreated control mice. Systemically, circulating B cells and monocytes (CD115+Ly6C+) were reduced in pristane-treated Ifnlr1−/− mice. The present study supports a significant role for type III interferons in the pathogenesis of pristane-induced murine autoimmunity as well as in systemic and renal inflammation. Although the absence of type III interferon receptors does not completely prevent the development of autoantibodies, type III interferon signaling accelerates the development of autoimmunity and promotes a pro-inflammatory environment in autoimmune-prone hosts.

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. New findings indicate that IFN-λ (type III IFN) has a non-redundant role in antiviral, antifungal and antiprotozoal defences of mucosal barriers that differs in several aspects from the functions of IFN-α and IFN-β (type I IFNs).

Previous work showed that interferon-λ (IFN-λ) can trigger the synthesis of thymic stromal lymphopoietin (TSLP) by specialized epithelial cells in the upper airways of mice, thereby improving the performance of intranasally administered influenza vaccines. Here we demonstrate that protein-only influenza vaccines containing either IFN-λ or TSLP boosted antigen-specific IgG1 and IgA responses and enhanced the resistance of mice to influenza virus challenge, irrespective of whether the vaccines were appliedviathe intranasal or the rectal route. TSLP receptor deficiency negatively influenced vaccine-induced antiviral immunity by impairing the migration of dendritic cells from the airways to the draining lymph nodes of immunized mice, thereby restraining follicular helper T cell and germinal center B cell responses. As previously observed during intranasal vaccination, the adjuvant effect of IFN-λ on a rectally administered influenza vaccine was no longer observed when TSLP receptor-deficient mice were used for immunization, highlighting the central role of the IFN-λ/TSLP axis for vaccine-induced antiviral immunity in the mucosa.

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.

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

Zoonotic avian influenza A virus (IAV) infections are rare. Sustained transmission of these IAVs between humans has not been observed, suggesting a role for host genes. We used whole-genome sequencing to compare avian IAV H7N9 patients with healthy controls and observed a strong association between H7N9 infection and rare, heterozygous single-nucleotide variants in the

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.

International audience; 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.

The commensal microbiota regulates susceptibility to enteric pathogens by fine-tuning mucosal innate immune responses, but how susceptibility to enteric viruses is shaped by the microbiota remains incompletely understood. Past reports have indicated that commensal bacteria may either promote or repress rotavirus replication in the small intestine of mice. We now report that rotavirus replicated more efficiently in the intestines of germ-free and antibiotic-treated mice compared to animals with an unmodified microbiota. Antibiotic treatment also facilitated rotavirus replication in type I and type III interferon (IFN) receptor-deficient mice, revealing IFN-independent proviral effects. Expression of interleukin-22 (IL-22) was strongly diminished in the intestine of antibiotic-treated mice. Treatment with exogenous IL-22 blocked rotavirus replication in microbiota-depleted wild-type and Stat1-/- mice, demonstrating that the antiviral effect of IL-22 in animals with altered microbiome is not dependent on IFN signaling. In antibiotic-treated animals, IL-22-induced a specific set of genes including Fut2, encoding fucosyl-transferase 2 that participates in the biosynthesis of fucosylated glycans which can mediate rotavirus binding. Interestingly, IL-22 also blocked rotavirus replication in antibiotic-treated Fut2-/- mice. Furthermore, IL-22 inhibited rotavirus replication in antibiotic-treated mice lacking key molecules of the necroptosis or pyroptosis pathways of programmed cell death. Taken together, our results demonstrate that IL-22 determines rotavirus susceptibility of antibiotic-treated mice, yet the IL-22-induced effector molecules conferring rotavirus resistance remain elusive.

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.

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.

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 (IkkβΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/β signaling-(IfnarΔHep), or interferon-α/β signaling in myeloid cells-(IfnarΔMyel) 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 IkkβΔHep 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 IkkβΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IfnarΔHep mice, whereas IfnarΔMyel 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.

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.

Type I and type III interferons (IFN) 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.IMPORTANCE Interferons can shape antiviral immune responses, but it is not understood well 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. By 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.

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. The role of IFN-λ in adaptive immunity is not well characterized. Staeheli and colleagues show that in the lungs, IFN-λ elicits production of the cytokine TSLP from M cells and that this in turn is essential for effective adaptive immunity and control of infection with influenza virus.

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.

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.

Flu immunity shows its age As we age, our immune systems change; in many ways not for the better. For instance, the elderly account for 90% of influenza deaths annually. Pillai et al. now report that influenza-infected human monocytes, a type of immune cell, exhibit reduced antiviral activity. In influenza-infected mice, two innate immune sensing pathways work together to promote antiviral immunity to influenza. Mice lacking antiviral immunity (similar to the situation in elderly people) had elevated bacterial burdens in their lungs and increased inflammatory responses, which both contributed to their increased susceptibility to influenza. Science , this issue p. 463

Interferon beta (IFN-β) is a key component of cellular innate immunity in mammals, and it constitutes the first line of defense during viral infection. Studies with cultured cells previously showed that almost all nucleated cells are able to produce IFN-β to various extents, but information about the in vivo sources of IFN-β remains incomplete. By applying immunohistochemistry and employing conditional-reporter mice that express firefly luciferase under the control of the IFN-β promoter in either all or only distinct cell types, we found that astrocytes are the main producers of IFN-β after infection of the brain with diverse neurotropic viruses, including rabies virus, Theiler's murine encephalomyelitis virus, and vesicular stomatitis virus. Analysis of a panel of knockout mouse strains revealed that sensing of viral components via both RIG-I-like helicases and Toll-like receptors contributes to IFN induction in the infected brain. A genetic approach to permanently mark rabies virus-infected cells in the brain showed that a substantial number of astrocytes became labeled and, therefore, must have been infected by the virus at least transiently. Thus, our results strongly indicate that abortive viral infection of astrocytes can trigger pattern recognition receptor signaling events which result in secretion of IFN-β that confers antiviral protection. IMPORTANCE Previous work indicated that astrocytes are the main producers of IFN after viral infection of the central nervous system (CNS), but it remained unclear how astrocytes might sense those viruses which preferentially replicate in neurons. We have now shown that virus sensing by both RIG-I-like helicases and Toll-like receptors is involved. Our results further demonstrate that astrocytes get infected in a nonproductive manner under these conditions, indicating that abortive infection of astrocytes plays a previously unappreciated role in the innate antiviral defenses of the CNS.

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., Author summary Infection with viruses and other pathogens requires appropriate cellular countermeasures, which involve swift and accurate adaptation of gene expression profiles. mRNAs encoding for immune-regulatory and effector proteins need to be transported into the cytoplasm in order to generate proteins necessary to fight the pathogen. Here we show that this process requires proper functionality of the Nuclear cap protein 3 (NCBP3), a protein recently identified to contribute to an alternative mRNA cap-binding complex. An Ncbp3-deficient mouse model allowed higher virus growth in vitro and showed high susceptibility to influenza A virus challenge in vivo. While NCBP3-deficient cells were able to transcriptionally upregulate cytokine mRNAs, generation of cytokines was significantly reduced in the absence of NCBP3. Our data shows a non-redundant function of NCBP3 and the alternative cap-binding complex in antiviral responses. More broadly, this work demonstrates a yet unappreciated aspect of post-transcriptional gene regulation.

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., Smac mimetics sensitize cancer cells to the extrinsic cell death pathway and stimulate anti-tumour immunity. In this study, the authors show that Smac mimetics can synergize with immune checkpoint inhibitors to control tumour growth in mouse cancer models, including aggressive CNS tumours, in a cytotoxic CD8+ T-cell- and TNFα-dependent manner.

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.

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 Fcgr1-/-Fcgr3-/- 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 naive contacts.