Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.

B cell depletion impairs vaccination-induced CD8+ T cell responses in a type I interferon-dependent manner.

OBJECTIVES: The monoclonal anti-CD20 antibody rituximab is frequently applied in the treatment of lymphoma as well as autoimmune diseases and confers efficient depletion of recirculating B cells. Correspondingly, B cell-depleted patients barely mount de novo antibody responses during infections or vaccinations. Therefore, efficient immune responses of B cell-depleted patients largely depend on protective T cell responses. METHODS: CD8+ T cell expansion was studied in rituximab-treated rheumatoid arthritis (RA) patients and B cell-deficient mice on vaccination/infection with different vaccines/pathogens. RESULTS: Rituximab-treated RA patients vaccinated with Influvac showed reduced expansion of influenza-specific CD8+ T cells when compared with healthy controls. Moreover, B cell-deficient JHT mice infected with mouse-adapted Influenza or modified vaccinia virus Ankara showed less vigorous expansion of virus-specific CD8+ T cells than wild type mice. Of note, JHT mice do not have an intrinsic impairment of CD8+ T cell expansion, since infection with vaccinia virus induced similar T cell expansion in JHT and wild type mice. Direct type I interferon receptor signalling of B cells was necessary to induce several chemokines in B cells and to support T cell help by enhancing the expression of MHC-I. CONCLUSIONS: Depending on the stimulus, B cells can modulate CD8+ T cell responses. Thus, B cell depletion causes a deficiency of de novo antibody responses and affects the efficacy of cellular response including cytotoxic T cells. The choice of the appropriate vaccine to vaccinate B cell-depleted patients has to be re-evaluated in order to efficiently induce protective CD8+ T cell responses.

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis.

BACKGROUND & AIM: Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation. METHODS: Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-ß induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1+/gfp mice revealed the origin of reconstituted KC. Finally, mixed bone marrow chimeric mice were studied to specifically analyze the effect of IFNAR triggering on liver infiltrating monocytes. RESULTS: VACV infection induced local IFN-ß responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution. CONCLUSION: Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis. LAY SUMMARY: Viral infection can cause fulminant hepatitis, which in turn is a major cause of acute liver failure. Herein, we aimed to study the role of type 1 interferon responses in acute viral hepatitis. We identified that during infection with DNA-encoded viruses, type 1 interferon receptor triggering of blood monocytes delays the development of monocyte-derived Kupffer cells. This points to new therapeutic strategies for acute viral hepatitis.

Proliferation status defines functional properties of endothelial cells.

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

Interferon regulatory factor-1 protects from fatal neurotropic infection with vesicular stomatitis virus by specific inhibition of viral replication in neurons.

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1-/- mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1-/- mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.

Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency.

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNß blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNß is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNß. Finally, IFNß prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNß is consistent with the establishment of CMV latency.

Uncoupling of the dynamics of host-pathogen interaction uncovers new mechanisms of viral interferon antagonism at the single-cell level.

Antiviral defence in mammals is mediated through type-I interferons (IFNs). Viruses antagonise this process through expression of IFN antagonist proteins (IAPs). Understanding and modelling of viral escape mechanisms and the dynamics of IAP action has the potential to facilitate the development of specific and safe drugs. Here, we describe the dynamics of interference by selected viral IAPs, NS1 from Influenza A virus and NS3/4A from Hepatitis C virus. We used Tet-inducible IAP gene expression to uncouple this process from virus-driven dynamics. Stochastic activation of the IFN-ß gene required the use of single-cell live imaging to define the efficacy of the inhibitors during the virus-induced signalling processes. We found significant correlation between the onset of IAP expression and halted IFN-ß expression in cells where IFN-ß induction had already occurred. These data indicate that IAPs not only prevent antiviral signalling prior to IFN-ß induction, but can also stop the antiviral response even after it has been activated. We found reduced NF-κB activation to be the underlying mechanism by which activated IFN expression can be blocked. This work demonstrates a new mechanism by which viruses can antagonise the IFN response.

Enhanced in vivo efficacy of a type I interferon superagonist with extended plasma half-life in a mouse model of multiple sclerosis.

IFNß is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNß. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35-55-induced experimental autoimmune encephalomyelitis compared with IFNß, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.

A comparative study of matrix metalloproteinase and aggrecanase mediated release of latent cytokines at arthritic joints.

BACKGROUND: Latent cytokines are engineered by fusing the latency associated peptide (LAP) derived from transforming growth factor-ß (TGF-ß) with the therapeutic cytokine, in this case interferon-ß (IFN-ß), via an inflammation-specific matrix metalloproteinase (MMP) cleavage site. OBJECTIVES: To demonstrate latency and specific delivery in vivo and to compare therapeutic efficacy of aggrecanase-mediated release of latent IFN-ß in arthritic joints to the original MMP-specific release. METHODS: Recombinant fusion proteins with MMP, aggrecanase or devoid of cleavage site were expressed in CHO cells, purified and characterised in vitro by Western blotting and anti-viral protection assays. Therapeutic efficacy and half-life were assessed in vivo using the mouse collagen-induced arthritis model (CIA) of rheumatoid arthritis and a model of acute paw inflammation, respectively. Transgenic mice with an IFN-regulated luciferase gene were used to assess latency in vivo and targeted delivery to sites of disease. RESULTS: Efficient localised delivery of IFN-ß to inflamed paws, with low levels of systemic delivery, was demonstrated in transgenic mice using latent IFN-ß. Engineering of latent IFN-ß with an aggrecanase-sensitive cleavage site resulted in efficient cleavage by ADAMTS-4, ADAMTS-5 and synovial fluid from arthritic patients, with an extended half-life similar to the MMP-specific molecule and greater therapeutic efficacy in the CIA model. CONCLUSIONS: Latent cytokines require cleavage in vivo for therapeutic efficacy, and they are delivered in a dose dependent fashion only to arthritic joints. The aggrecanase-specific cleavage site is a viable alternative to the MMP cleavage site for the targeting of latent cytokines to arthritic joints.

Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response.

The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-ß expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-ß expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.

A universal microfluidic approach for integrated analysis of temporal homocellular and heterocellular signaling and migration dynamics.

Microfluidics offers precise and dynamic control of microenvironments for the study of temporal cellular responses. However, recent research focusing solely on either homocellular (single-cell, population) or heterocellular response may yield insufficient output, which possibly leads to partial comprehension about the underlying mechanisms of signaling events and corresponding cellular behaviors. Here, a universal microfluidic approach is developed for integrated analysis of temporal signaling and cell migration dynamics in multiple cellular contexts (single-cell, population and coculture). This approach allows to confine the desired number or mixture of specific cell sample types in a single device. Precise single cell seeding was achieved manually with bidirectional controllability. Coupled with time-lapse imaging, temporal cellular responses can be observed with single-cell resolution. Using NIH3T3 cells stably expressing signal transducer and activator of transcription 1/2 (STAT1/2) activity biosensors, temporal STAT1/2 activation and cell migration dynamics were explored in isolated single cells, populations and cocultures stimulated with temporal inputs, such as single-pulse and continuous signals of interferon γ (IFNγ) or lipopolysaccharide (LPS). We demonstrate distinct dynamic responses of fibroblasts in different cellular contexts. Our presented approach facilitates a multi-dimensional understanding of STAT signaling and corresponding migration behaviors.

Type I Interferon Signaling Controls Gammaherpesvirus Latency In Vivo.

Gammaherpesviruses, such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are important human pathogens involved in lymphoproliferative disorders and tumorigenesis. Herpesvirus infections are characterized by a biphasic cycle comprised of an acute phase with lytic replication and a latent state. Murine gammaherpesvirus 68 (MHV-68) is a well-established model for the study of lytic and latent life cycles in the mouse. We investigated the interplay between the type I interferon (IFN)-mediated innate immune response and MHV-68 latency using sensitive bioluminescent reporter mice. Adoptive transfer of latently infected splenocytes into type I IFN receptor-deficient mice led to a loss of latency control. This was revealed by robust viral propagation and dissemination of MHV-68, which coincided with type I IFN reporter induction. Despite MHV-68 latency control by IFN, the continuous low-level cell-to-cell transmission of MHV-68 was detected in the presence of IFN signaling, indicating that IFN cannot fully prevent viral dissemination during latency. Moreover, impaired type I IFN signaling in latently infected splenocytes increased the risk of virus reactivation, demonstrating that IFN directly controls MHV-68 latency in infected cells. Overall, our data show that locally constrained type I IFN responses control the cellular reservoir of latency, as well as the distribution of latent infection to potential new target cells.

Temporal and spatial resolution of type I and III interferon responses in vivo.

Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.

Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1.

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE-luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.

Defective interferon amplification and impaired host responses against influenza virus in obese mice.

OBJECTIVE: Obesity is a major risk factor that increases morbidity and mortality upon infection. Although type I and type III interferon (IFN)-induced innate immune responses represent the first line of defense against viral infections, their functionality in the context of metabolic disorders remains largely obscure. This study aimed to investigate IFN responses upon respiratory viral infection in obese mice. METHODS: The activation of IFNs as well as IFN regulatory factors (IRFs) upon H3N2 influenza infection in mice upon high-fat-diet feeding was investigated. RESULTS: Influenza infection of obese mice was characterized by higher mortalities. In-depth analysis revealed impaired induction of both type I and type III IFNs as well as markedly reduced IFN responses. Notably, it was found that IRF7 gene expression in obese animals was reduced in homeostasis, and its induction by the virus was strongly attenuated. CONCLUSIONS: The results suggest that the attenuated IRF7 expression and induction are responsible for the reduced expression levels of type I and III IFNs and, thus, for the higher susceptibility and severity of respiratory infections in obese mice.

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.

Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation.

BACKGROUND: Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence. RESULTS: Coexpression of alphaT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with alphaT2ib indicated interaction of alphaT2ib with its cognate antigen within cells. alphaT2ib inhibited NF-kappaB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding alphaT2ib into HEK293 cells demonstrated high efficiency of the TLR2-alphaT2ib interaction. The alphaT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-alphaT2ib. Transduction with AdValphaT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFalpha mRNA accumulation, as well as TNFalpha translation and release by macrophages were largely abrogated upon transduction of alphaT2ib. alphaT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdValphaT2ib. Systemic transduction applying AdValphaT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. CONCLUSION: The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of alphaT2ib in microbial or viral infections.

Innate signalling molecules as genetic adjuvants do not alter the efficacy of a DNA-based influenza A vaccine.

In respect to the heterogeneity among influenza A virus strains and the shortcomings of current vaccination programs, there is a huge interest in the development of alternative vaccines that provide a broader and more long-lasting protection. Gene-based approaches are considered as promising candidates for such flu vaccines. In our study, innate signalling molecules from the RIG-I and the NALP3 pathways were evaluated as genetic adjuvants in intramuscular DNA immunizations. Plasmids encoding a constitutive active form of RIG-I (cRIG-I), IPS-1, IL-1ß, or IL-18 were co-administered with plasmids encoding the hemagglutinin and nucleoprotein derived from H1N1/Puerto Rico/8/1934 via electroporation in BALB/c mice. Immunogenicity was analysed in detail and efficacy was demonstrated in homologous and heterologous influenza challenge experiments. Although the biological activities of the adjuvants have been confirmed by in vitro reporter assays, their single or combined inclusion in the vaccine did not result in superior vaccine efficacy. With the exception of significantly increased levels of antigen-specific IgG1 after the co-administration of IL-1ß, there were only minor alterations concerning the immunogenicity. Since DNA electroporation alone induced substantial inflammation at the injection site, as demonstrated in this study using Mx2-Luc reporter mice, it might override the adjuvants´ contribution to the inflammatory microenvironment and thereby minimizes the influence on the immunogenicity. Taken together, the DNA immunization was protective against subsequent challenge infections but could not be further improved by the genetic adjuvants analysed in this study.

Innate immune responses in NF-kappaB-repressing factor-deficient mice.

NF-kappaB-repressing factor (NRF) is a transcriptional silencer protein that specifically counteracts the basal activity of several NF-kappaB-dependent promoters by direct binding to specific neighboring DNA sequences. In cell culture experiments, the reduction of NRF mRNA leads to a derepression of beta interferon, interleukin-8, and inducible nitric oxide synthase transcription. The X chromosome-located single-copy NRF gene is ubiquitously expressed and encodes a protein of 690 amino acids. The N-terminal part contains a nuclear localization signal, the DNA-binding domain, and the NF-kappaB-repressing domain, while the C-terminal part is responsible for double-stranded RNA binding and nucleolar localization. To study the function of NRF in a systemic context, transgenic mice lacking the NRF gene were created. Against predictions from in vitro experiments, mice with a deletion of the NRF gene are viable and have a phenotype that is indistinguishable from wild-type mice, even after challenge with different pathogens. The data hint towards an unexpected functional redundancy of NRF.