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

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.

Viral recognition and the antiviral interferon response.

Interferons (IFNs) are antiviral cytokines that play a key role in the innate immune response to viral infections. In response to viral stimuli, cells produce and release interferons, which then act on neighboring cells to induce the transcription of hundreds of genes. Many of these gene products either combat the viral infection directly, e.g., by interfering with viral replication, or help shape the following immune response. Here, we review how viral recognition leads to the production of different types of IFNs and how this production differs in spatial and temporal manners. We then continue to describe how these IFNs play different roles in the ensuing immune response depending on when and where they are produced or act during an infection.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity.

The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na+ -dependent Cl- - and K+ -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+ , K+ , and 2 Cl- ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl- -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

Effective Interferon Lambda Treatment Regimen To Control Lethal MERS-CoV Infection in Mice.

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

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

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.

The IFNL4 Gene Is a Noncanonical Interferon Gene with a Unique but Evolutionarily Conserved Regulation.

Interferon lambda 4 (IFN-λ4) is a recently identified enigmatic member of the interferon (IFN) lambda family. Genetic data suggest that the IFNL4 gene acts in a proviral and anti-inflammatory manner in patients. However, the protein is indistinguishable in vitro from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 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 IFNL4 This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans.IMPORTANCE Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner, as patients with a nonfunctional IFNL4 gene exhibit increased clearance of hepatitis C virus (HCV) but also increased liver inflammation. This suggests that the IFNL4 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 IFNL4 gene and its gene product, interferon lambda 4 (IFN-λ4). We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 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 IFNL4 gene is related to its noncanonical transcriptional regulation.

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

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/ß) 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 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.

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.

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.

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

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-1ß 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-1ß 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.

Length dependent activation of OAS proteins by dsRNA.

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.

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

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.

Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling.

The oligoadenylate synthetase (OAS) enzymes are cytoplasmic dsRNA sensors belonging to the antiviral innate immune system. Upon binding to viral dsRNA, the OAS enzymes synthesize 2'-5' linked oligoadenylates (2-5As) that initiate an RNA decay pathway to impair viral replication. The human OAS-like (OASL) protein, however, does not harbor the catalytic activity required for synthesizing 2-5As and differs from the other human OAS family members by having two C-terminal ubiquitin-like domains. In spite of its lack of enzymatic activity, human OASL possesses antiviral activity. It was recently demonstrated that the ubiquitin-like domains of OASL could substitute for K63-linked poly-ubiquitin and interact with the CARDs of RIG-I and thereby enhance RIG-I signaling. However, the role of the OAS-like domain of OASL remains unclear. Here we present the crystal structure of the OAS-like domain, which shows a striking similarity with activated OAS1. Furthermore, the structure of the OAS-like domain shows that OASL has a dsRNA binding groove. We demonstrate that the OAS-like domain can bind dsRNA and that mutating key residues in the dsRNA binding site is detrimental to the RIG-I signaling enhancement. Hence, binding to dsRNA is an important feature of OASL that is required for enhancing RIG-I signaling.

The 2'-5'-oligoadenylate synthetase 3 enzyme potently synthesizes the 2'-5'-oligoadenylates required for RNase L activation.

UNLABELLED: The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS. IMPORTANCE: We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.

Mapping of Chikungunya virus interactions with host proteins identified nsP2 as a highly connected viral component.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus.

PD-L1 and PD-L2 immune checkpoint protein induction by type III interferon in non-small cell lung cancer cells.

INTRODUCTION: Despite the clinical success of PD-1/PD-1-ligand immunotherapy in non-small cell lung cancer (NSCLC), the appearance of primary and acquired therapy resistance is a major challenge reflecting that the mechanisms regulating the expression of the PD-1-ligands PD-L1 and PD-L2 are not fully explored. Type I and II interferons (IFNs) induce PD-L1 and PD-L2 expression. Here, we examined if PD-L1 and PD-L2 expression also can be induced by type III IFN, IFN-λ, which is peculiarly important for airway epithelial surfaces. METHODS: In silico mRNA expression analysis of PD-L1 (CD274), PD-L2 (PDCD1LG2), and IFN- λ signaling signature genes in NSCLC tumors and cell lines was performed using RNA sequencing expression data from TCGA, OncoSG, and DepMap portals. IFN-λ-mediated induction of PD-L1 and PD-L2 expression in NSCLC cell lines was examined by real-time quantitative polymerase chain reaction and flow cytometry. RESULTS: IFNL genes encoding IFN- λ variants are expressed in the majority of NSCLC tumors and cell lines along with the IFNLR1 and IL10R2 genes encoding the IFN-λ receptor subunits. The expression of PD-L1 and PD-L2 mRNA is higher in NSCLC tumors with IFNL mRNA expression compared to tumors without IFNL expression. In the NSCLC cell line HCC827, stimulation with IFN-λ induced both an increase in PD-L1 and PD-L2 mRNA expression and cell surface abundance of the corresponding proteins. In the NSCLC cell line A427, displaying a low basal expression of PD-L1 and PD-L2 mRNA and corresponding proteins, stimulation with IFN-λ resulted in an induction of the former. CONCLUSION: The type III IFN, IFN- λ, is capable of inducing PD-L1 and PD-L2 expression, at least in some NSCLC cells, and this regulation will need acknowledgment in the development of new diagnostic procedures, such as gene expression signature profiles, to improve PD-1/PD-1-ligand immunotherapy in NSCLC.

Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus.

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

Selection of a novel and highly specific tumor necrosis factor alpha (TNFalpha) antagonist: insight from the crystal structure of the antagonist-TNFalpha complex.

Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.

Interferon-λ Improves the Efficacy of Intranasally or Rectally Administered Influenza Subunit Vaccines by a Thymic Stromal Lymphopoietin-Dependent Mechanism.

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 applied via the 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.