Coxsackievirus counters the host innate immune response by blocking type III interferon expression.

Type I IFNs play an important role in the immune response to enterovirus infections. Their importance is underscored by observations showing that many enteroviruses including coxsackie B viruses (CVBs) have developed strategies to block type I IFN production. Recent studies have highlighted a role for the type III IFNs (also called IFNλs) in reducing permissiveness to infections with enteric viruses including coxsackievirus. However, whether or not CVBs have measures to evade the effects of type III IFNs remains unknown. By combining virus infection studies and different modes of administrating the dsRNA mimic poly I : C, we discovered that CVBs target both TLR3- and MDA5/RIG-I-mediated type III IFN expression. Consistent with this, the cellular protein expression levels of the signal transduction proteins TRIF and IPS1 were reduced and no hyperphosphorylation of IRF-3 was observed following infection with the virus. Notably, decreased expression of full-length TRIF and IPS1 and the appearance of cleavage products was observed upon both CVB3 infection and in cellular protein extracts incubated with recombinant 2Apro, indicating an important role for the viral protease in subverting the cellular immune system. Collectively, our study reveals that CVBs block the expression of type III IFNs, and that this is achieved by a similar mechanism as the virus uses to block type I IFN production. We also demonstrate that the virus blocks several intracellular viral recognition pathways of importance for both type I and III IFN production. The simultaneous targeting of numerous arms of the host immune response may be required for successful viral replication and dissemination.

Cellular localization and effects of ectopically expressed hepatitis A virus proteins 2B, 2C, 3A and their intermediates 2BC, 3AB and 3ABC.

In the course of hepatitis A virus (HAV) infections, the seven nonstructural proteins and their intermediates are barely detectable. Therefore, little is known about their functions and mechanisms of action. Ectopic expression of the presumably membrane-associated proteins 2B, 2C, 3A and their intermediates 2BC, 3AB and 3ABC allowed the intracellular localization of these proteins and their possible function during the replication cycle of HAV to be investigated. In this study, we used rhesus monkey kidney cells, which are commonly used for cell culture experiments, and human liver cells, which are the natural target cells. We detected specific associations of these proteins with distinct membrane compartments and the cytoskeleton, different morphological alterations of the respective structures, and specific effects on cellular functions. Besides comparable findings in both cell lines used with regard to localization and effects of the proteins examined, we also found distinct differences. The data obtained identify so far undocumented interactions with and effects of the HAV proteins investigated on cellular components, which may reflect unknown aspects of the interaction of HAV with the host cell, for example the modification of the ERGIC (ER-Golgi intermediate compartment) structure, an interaction with lipid droplets and lysosomes, and inhibition of the classical secretory pathway.

A preclinical study on the efficacy and safety of a new vaccine against Coxsackievirus B1 reveals no risk for accelerated diabetes development in mouse models.

AIMS/HYPOTHESIS: Enterovirus infections have been implicated in the aetiology of autoimmune type 1 diabetes. A vaccine could be used to test the causal relationship between enterovirus infections and diabetes development. However, the development of a vaccine against a virus suspected to induce an autoimmune disease is challenging, since the vaccine itself might trigger autoimmunity. Another challenge is to select the enterovirus serotypes to target with a vaccine. Here we aimed to evaluate the function and autoimmune safety of a novel non-adjuvanted prototype vaccine to Coxsackievirus serotype B1 (CVB1), a member of the enterovirus genus. METHODS: A formalin-inactivated CVB1 vaccine was developed and tested for its immunogenicity and safety in BALB/c and NOD mice. Prediabetic NOD mice were vaccinated, infected with CVB1 or mock-treated to compare the effect on diabetes development. RESULTS: Vaccinated mice produced high titres of CVB1-neutralising antibodies without signs of vaccine-related side effects. Vaccinated mice challenged with CVB1 had significantly reduced levels of replicating virus in their blood and the pancreas. Prediabetic NOD mice demonstrated an accelerated onset of diabetes upon CVB1 infection whereas no accelerated disease manifestation or increased production of insulin autoantibodies was observed in vaccinated mice. CONCLUSIONS/INTERPRETATION: We conclude that the prototype vaccine is safe and confers protection from infection without accelerating diabetes development in mice. These results encourage the development of a multivalent enterovirus vaccine for human use, which could be used to determine whether enterovirus infections trigger beta cell autoimmunity and type 1 diabetes in humans.

Inhibition of Type III Interferon Expression in Intestinal Epithelial Cells-A Strategy Used by Coxsackie B Virus to Evade the Host's Innate Immune Response at the Primary Site of Infection?

Increasing evidence highlights the importance of the antiviral activities of the type III interferons (IFNλs; IL-28A, IL-28B, IL29, and IFNλ4) in the intestine. However, many viruses have developed strategies to counteract these defense mechanisms by preventing the production of IFNs. Here we use infection models, a clinical virus isolate, and several molecular biology techniques to demonstrate that both type I and III IFNs induce an antiviral state and attenuate Coxsackievirus group B (CVB) replication in human intestinal epithelial cells (IECs). While treatment of IECs with a viral mimic (poly (I:C)) induced a robust expression of both type I and III IFNs, no such up-regulation was observed after CVB infection. The blunted IFN response was paralleled by a reduction in the abundance of proteins involved in the induction of interferon gene transcription, including TIR-domain-containing adapter-inducing interferon-ß (TRIF), mitochondrial antiviral-signaling protein (MAVS), and the global protein translation initiator eukaryotic translation initiation factor 4G (eIF4G). Taken together, this study highlights a potent anti-Coxsackieviral effect of both type I and III IFNs in cells located at the primary site of infection. Furthermore, we show for the first time that the production of type I and III IFNs in IECs is blocked by CVBs. These findings suggest that CVBs evade the host immune response in order to successfully infect the intestine.

Determining Laboratory Reference Values of TREC and KREC in Different Age Groups of Iranian Healthy Individuals.

Assessment of the number of T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) copies has been recently described as biomarkers of newly formed T and B cells respectively. In this study, we aimed to explore the effects of demographic variables including age, gender, weight, height and ethnicity on these two episomal DNA molecules. Second, for the first time in our country, we determined the reference values of TREC and KREC copy numbers in different age groups of Iranian healthy individuals as a threshold for identifying T cell and B cell lymphopenia. The TREC and KREC copy numbers were evaluated in 251 dried blood spot (DBS) samples from healthy volunteers (age range: 0-60 years). Six primary immunodeficiency (PID) patients were included as disease controls. TREC and KREC copies were markedly reduced with increasing age. Although the levels of TREC and KREC were higher in females than males, this difference did not reach statistical significance. These findings suggest that demographic variables including age should be considered for interpretation results of the TREC/KREC assay.

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms.

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2Apro and 3Cpro), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2Apro cause direct damage to the infected heart and tools to investigate 2Apro and 3Cpro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2Apro and 3Cpro; Two monoclonal 2Apro antibodies and one 3Cpro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3Cpro antibody detects all of the EV species B (EV-B) viruses tested and that the 2Apro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2Apro and 3Cpro, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.

An IFIH1 gene polymorphism associated with risk for autoimmunity regulates canonical antiviral defence pathways in Coxsackievirus infected human pancreatic islets.

The IFIH1 gene encodes the pattern recognition receptor MDA5. A common polymorphism in IFIH1 (rs1990760, A946T) confers increased risk for autoimmune disease, including type 1-diabetes (T1D). Coxsackievirus infections are linked to T1D and cause beta-cell damage in vitro. Here we demonstrate that the rs1990760 polymorphism regulates the interferon (IFN) signature expressed by human pancreatic islets following Coxsackievirus infection. A strong IFN signature was associated with high expression of IFNλ1 and IFNλ2, linking rs1990760 to the expression of type III IFNs. In the high-responding genotype, IRF-1 expression correlated with that of type III IFN, suggesting a positive-feedback on type III IFN transcription. In summary, our study uncovers an influence of rs1990760 on the canonical effector function of MDA5 in response to an acute infection of primary human parenchymal cells with a clinically relevant virus linked to human T1D. It also highlights a previously unrecognized connection between the rs1990760 polymorphism and the expression level of type III IFNs.

XOMA 052, an anti-IL-1{beta} monoclonal antibody, improves glucose control and {beta}-cell function in the diet-induced obesity mouse model.

Recent evidence suggests that IL-1beta-mediated glucotoxicity plays a critical role in type 2 diabetes mellitus. Although previous work has shown that inhibiting IL-1beta can lead to improvements in glucose control and beta-cell function, we hypothesized that more efficient targeting of IL-1beta with a novel monoclonal antibody, XOMA 052, would reveal an effect on additional parameters affecting metabolic disease. In the diet-induced obesity model, XOMA 052 was administered to mice fed either normal or high-fat diet (HFD) for up to 19 wk. XOMA 052 was administered as a prophylactic treatment or as a therapy. Mice were analyzed for glucose tolerance, insulin tolerance, insulin secretion, and lipid profile. In addition, the pancreata were analyzed for beta-cell apoptosis, proliferation, and beta-cell mass. Mice on HFD exhibited elevated glucose and glycated hemoglobin levels, impaired glucose tolerance and insulin secretion, and elevated lipid profile, which were prevented by XOMA 052. XOMA 052 also reduced beta-cell apoptosis and increased beta-cell proliferation. XOMA 052 maintained the HFD-induced compensatory increase in beta-cell mass, while also preventing the loss in beta-cell mass seen with extended HFD feeding. Analysis of fasting insulin and glucose levels suggests that XOMA 052 prevented HFD-induced insulin resistance. These studies provide new evidence that targeting IL-1beta in vivo could improve insulin sensitivity and lead to beta-cell sparing. This is in addition to previously reported benefits on glycemic control. Taken together, the data presented suggest that XOMA 052 could be effective for treating many aspects of type 2 diabetes mellitus.

CXCL10- a path to ß-cell death.

The ability of the ß-cells to control blood glucose levels depends on their function and mass. In both, type 1 and type 2 diabetes mellitus the main processes leading to ß-cell failure are apoptosis and loss of function. Many studies demonstrate how cytokines and chemokines have an active role in triggering the immune-response against the ß-cell population. In a recent study we have identified that the chemokine CXCL10 may play an active role in triggering ß-cell destruction.  We have identified the Toll like receptor 4 as the receptor for CXCL10 and as new pathway for the induction of ß-cell apoptosis. Our findings may open new therapeutic approaches to fight onset and progression of the disease.