Meleri Jones

Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression

ABSTRACT Alpha/beta interferon (IFN-α/β) is a key mediator of innate antiviral responses but has little effect on the established replication of dengue viruses, which are mosquito-borne flaviviruses of immense global health importance. Understanding how the IFN system is inhibited in dengue virus-infected cells would provide critical insights into disease pathogenesis. In a recent study analyzing the ability of individual dengue virus-encoded proteins to antagonize the IFN response, nonstructural (NS) protein 4B and possibly NS2A and NS4A were identified as candidate IFN antagonists. In monkey cells, NS4B appeared to inhibit both the IFN-α/β and IFN-γ signal transduction pathways, which are distinct but overlapping (J. L. Munoz-Jordan, G. G. Sanchez-Burgos, M. Laurent-Rolle, and A. Garcia-Sastre, Proc. Natl. Acad. Sci. USA 100:14333-14338, 2003). For this study, we examined the effects of dengue virus on the human IFN system, using cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and that expressed all of the dengue virus nonstructural proteins together. We show here that in replicon-containing cells dengue virus RNA replication and the replication of encephalomyocarditis virus, an IFN-sensitive virus, are resistant to the antiviral effects of IFN-α. The presence of dengue virus replicons reduces global IFN-α-stimulated gene expression and specifically inhibits IFN-α but not IFN-γ signal transduction. In cells containing replicons or infected with dengue virus, we found reduced levels of signal transducer and activator of transcription 2 (STAT2), which is a key component of IFN-α but not IFN-γ signaling. Collectively, these data show that dengue virus is capable of subverting the human IFN response by down-regulating STAT2 expression.

Herpes Simplex Virus Type-1-Induced Activation of Myeloid Dendritic Cells: The Roles of Virus Cell Interaction and Paracrine Type I IFN Secretion

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-κB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.

Qualitatively distinct patterns of cytokines are released by human dendritic cells in response to different pathogens

Dendritic cells produce cytokines that regulate the class of the adaptive immune response. Microbial recognition is mediated, at least in part, by pattern recognition receptors such as Toll‐like receptors, which influence dendritic cell maturation. In humans it is not yet clear how intact pathogens modulate the developing immune response. To address the effects of intact pathogens on the maturation and effector functions of human dendritic cells, we investigated their responses to a number of microbial pathogens. We studied a range of micro‐organisms including Gram‐negative bacteria (Escherichia coli and Salmonella enterica sv. typhimurium), Gram‐positive cocci (Staphylococcus aureus) and atypical bacteria (Mycobacterium tuberculosis and Mycoplasma hominis) as well as the human protozoal parasite Trichomonas vaginalis. The micro‐organisms were fixed in formaldehyde to prevent replication whilst preserving surface morphology. All the pathogens induced similar up‐regulation of dendritic cell activation‐associated cell surface markers but there was a profound difference in the patterns of cytokines produced by the stimulated dendritic cells. Some pathogens (E. coli, Salmonella enterica sv. typhimurium and S. aureus) induced interleukin‐12 (IL‐12), IL‐10 and interferon‐α whereas others (M. tuberculosis, Mycoplasma hominis and T. vaginalis) induced only IL‐10. This differential effect was not altered by costimulation of the dendritic cells through CD40. These results support the notion that human dendritic cells are plastic in their response to microbial stimuli and that the nature of the pathogen dictates the response of the dendritic cell.

Development and validation of a "capture-fusion" model to study drug sensitivity of patient-derived hepatitis C.

Emerging therapies for chronic hepatitis C viral (HCV) infection involve inhibition of viral enzymes with drug combinations. Natural, or treatment‐induced, enzyme polymorphisms reduce efficacy. We developed a phenotyping assay to aid drug selection based on viral transfer from monocytes to hepatocytes. We studied HCV in monocytes from infected patients and developed a model in which patient‐derived HCV is “captured” by the cell line THP‐1 and replication assessed after fusion to hepatoma cells. We found that monocytes from HCV‐infected patients harbor virus that replicates when cells are fused to hepatocytes. THP‐1 cells incubated with infected sera capture HCV, which replicates when fused to hepatocytes. Inhibitable replication of all HCV genotypes was achieved (42 of 52 isolates). We measured sensitivity of telaprevir (TVR) and alisporivir (AVR) in different genotypes, and showed differences in 50% inhibitory concentration (IC50) correlating with clinical response (TVR IC50 for genotype (G)1 was 0.042 ± 0.003 vs. 0.117 ± 0.015 μM for G3, whereas AVR IC50 for G1 was 0.139 ± 0.013 vs. 0.044 ± 0.007 μM for G3). We tested TVR‐resistant viral isolates and identified changes in IC50. One patient with a poor clinical response to TVR and wild‐type viral sequence showed reduced TVR sensitivity in our assay. We studied samples from a 2‐week TVR monotherapy study in which 5 of 8 patients with G3 HCV did not respond whereas 3 of 8 patients did. The “capture‐fusion” assay correctly identified responders. Conclusion: The capture‐fusion model represents a promising new technique that may help identify appropriate treatment strategies for patients with chronic HCV infection. (Hepatology 2015;61:1192–1204)

SB 9200, a novel agonist of innate immunity, shows potent antiviral activity against resistant HCV variants

SB 9200 is a novel, first‐in‐class oral modulator of innate immunity that is believed to act via the activation of the RIG‐I and NOD2 pathways. SB 9200 has broad‐spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10. Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture‐fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct‐acting antiviral treatment, including viruses containing known NS5A resistance‐associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens.

Stage of disease in hepatitis B virus infection in Zambian adults is associated with large cell change but not well defined using classic biomarkers

Abstract Background Hepatocellular malignancy in young adults is a prominent feature of hepatitis B virus (HBV) infection in southern Africa. Here we report a cross-sectional study of liver pathology correlated with biomarkers in adults with HBV infection in Zambia. Methods We analysed liver biopsies from Zambian patients with persistent HBV infection. Results We analysed 104 patients with HBV infection and evidence of liver disease. We obtained liver biopsies from 53 adults; of these, 12 (23%) were hepatitis B e antigen seropositive. The genotype was evenly distributed between A and E. One biopsy showed malignancy. Stage was 3 or more in 11 of 52 (21%) biopsies free of malignancy and lobular inflammation was found in 50 (94%). Neither alanine aminotransferase (ALT) nor the γ-glutamyl transferase:platelet ratio (GPR) were correlated with the stage of disease but were correlated with total Ishak score (ρ=0.47, p=0.0004 and ρ=0.33, p=0.02, respectively). Large cell change was observed in 10 of 11 biopsies with fibrosis stage 3 or more and 16 of 41 with early disease (p=0.005). Serum α-fetoprotein was elevated, although still within the normal range, in patients with large cell change (median 3.6 [interquartile range {IQR} 1.6–5.1]) compared with those without (1.7 [IQR 1.0–2.8]; p=0.03). Neither ALT nor GPR predicted large cell change. Conclusions Large cell change was common in young HBV-infected adults in Zambia. Only serum α-fetoprotein was identified as a biomarker of this phenotype.

P0703 : Pre-treatment ribavirin sensitivity correlates with treatment outcome in genotype 3 HCV

Background and Aims: New therapies for chronic HCV infection have substantially increased rates of sustained virological response (SVR). However relapse after therapy remains a problem, especially in patients with cirrhosis. We have developed a novel capture- fusion assay to study patient-derived HCV (1). Here we demonstrate that this assay can identify pre-treatment sofosbuvir (SOF), interferon (IFN) and ribavirin (RBV) sensitivity in patients with G3 HCV, and RBV sensitivity correlates with treatment outcome. Methods: Archived pre-treatment sera were obtained from 10 G3 patients treated with pegIFN/RBV, 4 with SVR and 6 who relapsed, and from 4 G3 patients treated with SOF/RBV, 3 with SVR and 1 who relapsed. THP-1 cells were exposed to donor serum, fused with Huh7.5 cells and treated with SOF, IFN or RBV before qPCR assessment of HCV replication. Results are given as mean ± sem and p values were calculated using Mann Whitney U test. Results: No difference in pre-treatment IFN sensitivity was seen between patients with SVR and those who relapsed after pegIFN/RBV (IFN IC50 0.61±0.11 IU/mL for patients with SVR versus 0.55±0.09 IU/mL for relapse, p = 0.61). However pre-treatment isolates from patients with SVR were significantly more sensitive to RBV than those from patients who relapsed (ribavirin IC50 0.62±0.05 mM for patients with SVR versus 1.25±0.13 m Mf or relapse, p = 0.01). Amongst patients treated with SOF/RBV, pre- treatment SOF sensitivity was similar between patients with SVR and relapse (SOF IC50 0.036±0.016 mM for SVR versus 0.023 m Mf or the patient who relapsed). However pre-treatment RBV sensitivity appeared greater in patients with SVR than relapse (RBV IC50 0.377±0.037 mM for SVR versus 1.049 mM for the patient who relapsed).