Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice.

BACKGROUND: The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases. METHODS: TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C). RESULTS: There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFalpha were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy. CONCLUSION: These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

Synthesis and biological activity of heteroaryl 3-(1,1-dioxo-2H-(1,2,4)-benzothiadizin-3-yl)-4-hydroxy-2(1H)-quinolinone derivatives as hepatitis C virus NS5B polymerase inhibitors.

Modification of the benzo rings of 3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones into heteroaromatic systems was investigated to enhance physicochemical properties and potency profile of this class of inhibitors. The synthesis and biological activity of the derived compounds is discussed.

Substituted benzothiadizine inhibitors of Hepatitis C virus polymerase.

The synthesis and optimisation of HCV NS5B polymerase inhibitors with improved potency versus the existing compound 1 is described. Substitution in the benzothiadiazine portion of the molecule, furnishing improvement in potency in the high protein Replicon assay, is highlighted, culminating in the discovery of 12h, a highly potent oxyacetamide derivative.

Deleterious role of TLR3 during hyperoxia-induced acute lung injury.

RATIONALE: Acute respiratory distress syndrome (ARDS) manifests clinically as a consequence of septic and/or traumatic injury in the lung. Oxygen therapy remains a major therapeutic intervention in ARDS, but this can contribute further to lung damage. Patients with ARDS are highly susceptible to viral infection and it may be due to altered Toll-like receptor (TLR) expression. OBJECTIVES: To evaluate the role of TLR3 in ARDS. METHODS: TLR3 expression and signaling was determined in airway epithelial cells after in vitro hyperoxia challenge. Using a murine model of hyperoxia-induced lung injury, the role of TLR3 was determined using either TLR3-gene deficient mice or a specific neutralizing antibody directed to TLR3. MEASUREMENTS AND MAIN RESULTS: Increased TLR3 expression was observed in airway epithelial cells from patients with ARDS. Further, hyperoxic conditions alone were a major stimulus for increased TLR3 expression and activation in cultured human epithelial cells. Interestingly, TLR3(-/-) mice exhibited less acute lung injury, activation of apoptotic cascades, and extracellular matrix deposition after 5 days of 80% oxygen compared with wild-type (TLR3(+/+)) mice under the same conditions. Administration of a monoclonal anti-TLR3 antibody to TLR3(+/+) mice exposed to hyperoxic conditions likewise protected these mice from lung injury and inflammation. CONCLUSIONS: The potential for redundancy in function as well as cross-talk between distinct TLRs may indeed contribute to whether the inflammatory cascade can be effectively disrupted once signaling has been initiated. Together, these data show that TLR3 has a major role in the development of ARDS-like pathology in the absence of a viral pathogen.

Down modulation of human TLR3 function by a monoclonal antibody.

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-kappaB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.

Factors that affect in vitro measurement of the susceptibility of herpes simplex virus to nucleoside analogues.

OBJECTIVES: To identify factors that contribute to variability of HSV antiviral susceptibility breakpoints. METHODS: Acyclovir and penciclovir IC(50)'s for 12 HSV clinical isolates were measured in two laboratories using plaque reduction assay (PRA), an enzyme immunoassay (EIA)-based antigen reduction, and DNA hybridization on Vero, A549, MRC-5, HEL299 and HELG monolayers. Pair-wise comparisons were performed to evaluate variables including testing laboratory, technique, monolayer, and antiviral. The proportion of false results was analyzed using a conventional susceptibility IC(50) breakpoint of 2 microg/ml. RESULTS: Acyclovir-resistant HSV isolates were correctly identified by all methods. In contrast, there were 6-67% of susceptible isolates incorrectly characterized as drug-resistant. Variables associated with these errors included testing site, assay method, cell line and antiviral. A549, DNA hybridization, and penciclovir were associated with the highest IC(50)'s, whereas the PRA, EIA, and human fibroblast-monolayers provided the best differentiation between susceptible and resistant HSV isolates. CONCLUSIONS: The current recommendations to use a single discriminating value to define HSV resistance to nucleoside analogues can be problematic. False results are influenced in various degrees by the laboratory method, tissue culture and antivirals.

Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus.

To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies.

A pathway analysis of poly(I:C)-induced global gene expression change in human peripheral blood mononuclear cells.

To gain global pathway perspective of ex vivo viral infection models using human peripheral blood mononuclear cells (PBMCs), we conducted expression analysis on PBMCs of healthy donors. RNA samples were collected at 3 and 24 h after PBMCs were challenged with the Toll-like receptor-3 (TLR3) agonist polyinosinic acid-polycytidylic acid [poly(I:C)] and analyzed by internally developed cDNA microarrays and TaqMan PCR. Our results demonstrate that poly(I:C) challenge can elicit certain gene expression changes, similar to acute viral infection. Hierarchical clustering revealed distinct immediate early, early-to-late, and late gene regulation patterns. The early responses were innate immune responses that involve TLR3, the NF-kappaB-dependent pathway, and the IFN-stimulated pathway, whereas the late responses were mostly cell-mediated immune response that involve activation of cell adhesion, cell mobility, and phagocytosis. Overall, our results expanded the utilities of this ex vivo model, which could be used to screen molecules that can modulate viral stress-induced inflammation, in particular those mediated via TLRs.

Not-so-sweet sixteen: the role of IL-16 in infectious and immune-mediated inflammatory diseases.

Over the past two decades, our understanding of interleukin-16 (IL-16) has increased substantially. Initial studies characterizing IL-16 as a chemotactic cytokine (but not a chemokine) just scratched the surface of the unique properties of this cytokine. Since then, scientists have determined that IL-16 has a wide range of effects on cells, including upregulation of CD25, induction of cells to progress to the G(1) phase, inhibition of antigen- specific proliferation yet with retained antigen nonspecific proliferative properties, and discovery of a novel neuronal form with unique properties. Recently, a plethora of studies have implicated IL-16 in exacerbation of infectious, immune-mediated, and autoimmune inflammatory disorders, including atopic dermatitis, irritable bowel syndrome, systemic lupus erythematosus, neurodegenerative disorders, and viral infections. Herein, we review the body of evidence supporting a role for IL-16 in infectious and immune-mediated inflammatory disorders and explore the known and possible mechanism of actions in the numerous diseases.

3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones, potent inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function.

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32 degrees C, 37 degrees C, or 39.5 degrees C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.

Novel treatment options for infectious exacerbations.

Herein, we detail characteristics shared between two common therapeutic approaches, macrolide antibiotic therapy and systemic corticosteroids, in an attempt to propose an alternative treatment paradigm.

Multiple interactions within the hepatitis C virus RNA polymerase repress primer-dependent RNA synthesis.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) initiates RNA synthesis in vivo by a de novo mechanism. In vitro, however, the HCV RdRp can initiate de novo or extend from a primed template. A novel beta-loop near the RdRp active site was previously found to prevent the use of primed templates. We found that, in addition to the beta-loop, the C-terminal tail of the HCV RdRp and the de novo initiation GTP are required to exclude the use of primed-templates. GTP binding to the NTPi site of the HCV RdRp orchestrates the participation of other structures. The interactions of the beta-loop, C-terminal tail, and GTP provide an elegant solution to ensure de novo initiation of HCV RNA synthesis.

Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein.

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.

Modulating toll-like receptor signalling as a novel antiinfective approach.

The innate immune response against invading microorganisms results in the deployment of phagocytes, including macrophages and dendritic cells to recognize pathogen-associated molecular patterns. Activation of Toll-like receptors (TLRs) expressed on these cells is a critical step in the initiation of this response, triggering the production of pro- and antiinflammatory cytokines to dampen microbial pathogenesis. Importantly, TLR activation also mediates dendritic cell maturation, a critical step in bridging the innate and adaptive arms of the immune system. Balancing the role of TLRs as central mediators of overlapping signaling pathways, whether directly through ligand interactions or via secondary adaptor molecules, mandates exquisite specificity. Further, understanding the immunopharmacology of TLR cross-talk during infection may help to provide insight into innate immunity and the mechanisms of immune-response subversion by pathogens. The continual and rapid emergence of drug resistance to traditional antimicrobial agents highlights the medical need for new treatment approaches. Herein, the discovery and development of TLR agonist and antagonist therapies for infectious diseases as adjunct to, or in place of, conventional treatment paradigms is discussed.

What is disrupting IFN-alpha's antiviral activity?

Despite advances in treatment strategies for hepatitis C virus (HCV), a significant proportion of patients fail to achieve viral clearance following treatment with pegylated interferon (IFN)-alpha plus ribavirin. Many of these individuals show elevated levels of tumor necrosis factor (TNF)-alpha compared with normal controls, and recent data have implicated this cytokine in the negative regulation of IFN-alpha. Although a therapeutic opportunity for TNF-alpha antagonists might exist for reducing inflammation in chronic HCV disease, further exploration is required to identify the key mediators of responsiveness to IFN-alpha. In particular, the interplay should be clarified between host response factors [e.g. IFN-alpha, IFN-gamma, suppressor of cytokine signaling (SOCS), TNF-alpha and others] and pathogen-associated molecular patterns [PAMPs, e.g. lipopolysaccharide (LPS) and CpG DNA] in HCV disease; this information might guide future therapies aimed at improving IFN-alpha responsiveness.

Biomedicines to reduce inflammation but not viral load in chronic HCV--what's the sense?

Although cytokines and cytotoxic T lymphocytes (CTL) are among the predominant mechanisms of host defense against viral pathogens, they can induce an inflammatory response that often leads to tissue injury. Hepatitis C virus (HCV) infection, a major cause of liver-related disease, results in the induction of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), and CTL activity, followed by liver injury. Although inflammation facilitates the wound healing process, chronic persistence over several decades results in scar accumulation, fibrosis and often cirrhosis. This review summarizes biological data implicating a cause-and-effect relationship between TNF-alpha levels and the progression of fibrosis in chronic HCV infections, in contrast to the role of TNF-alpha in hepatitis B virus infections. Furthermore, an overview of therapeutic approaches to halting the inflammatory cascade in individuals with chronic HCV, including the use of agents to reduce the level of TNF-alpha, is presented.

Sequence requirements for the development of a chimeric HCV replicon system.

The hepatitis C virus (HCV) 3'nontranslated region (3'NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3'NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3'NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3'NTR supported stable replication suggesting that the interaction between the NS proteins and the 3'NTR may represent a critical determinant. Lastly, the type 1a 3'NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3'NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3'NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3'NTR sequences from relevant clinical samples.

Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility.

BACKGROUND: A number of in vitro assays are used to determine susceptibility of HSV to antiviral agents, but results from these in vitro assays do not necessarily correlate with treatment outcome. OBJECTIVES: A method with improved capability for identifying an isolate as acyclovir (ACV) or penciclovir (PCV) resistant when resistance is borderline could greatly improve the management of HSV disease. STUDY DESIGN: A comparative evaluation of four in vitro assays, plaque reduction (PRA), DNA hybridization, plating efficiency (PEA) and plaque autoradiography (PAR) was performed to accurately identify and measure resistance of a TK-altered clinical HSV isolate (HSV-1 N4) from a patient who was non-responsive to ACV treatment. Two established criteria for the prediction of antiviral resistance, IC(50)> or =2.0 microg/ml or an IC(50) greater than 10x above a sensitive virus IC(50), as well as testing in human (MRC-5) and nonhuman (Vero and CV-1 monkey kidney) cell lines were evaluated. RESULTS: The PRA and DNA hybridization assays accurately identified HSV-1 N4 as ACV(r) in human cells when using the 10x above sensitive virus IC(50) resistance criterion. Moreover, the PEA and PAR assays failed to classify HSV-1 N4 as drug resistant and indicate that these technologies alone are inadequate for identifying resistant virus. CONCLUSIONS: The data presented herein indicate that the PRA and DNA hybridization assays most accurately identified an otherwise borderline-resistant isolate as drug resistant: (i) when a sensitive virus is used within each individual assay as a control, (ii) when ACV and PCV susceptibility is evaluated in human cells, and (iii) when the 10x above sensitive IC(50) criterion is used to classify a virus as drug-resistant. Testing of additional clinical samples is warranted to further confirm these findings.

Intracellular hepatitis C virus RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) infection represents a significant health concern in over 170 million individuals worldwide. Recently, Huh7 cell-based hepatitis C virus replicon systems, which rely upon the expression and cooperation of viral nonstructural proteins to mediate replication of the entire hepatitis C virus genome, were shown to be useful for studying viral replication and antiviral agents. We report that expression of the viral RNA-dependent RNA polymerase (RdRp) in yeast cells, independent of other viral proteins, is necessary and sufficient for initiation of RNA synthesis in cis from 3'-nontranslated hepatitis C virus RNA. Furthermore, expression of the polymerase alone appears incapable of transcribing across the entire viral genome, most likely due to the secondary structure of the RNA. Other viral polypeptides, such as helicase, which are presumed to be present in the functional replicase complex, are predicted to facilitate RNA synthesis across highly structured regions.