ABSTRACT
The incidence of Clostridioides difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. Here we report on the development of mRNA-LNPs encoding camelid-derived VHH-based neutralizing agents (VNAs) targeting toxins A and/or B of C. difficile. In preclinical models, intravenous administration of the mRNA-LNPs provided serum VNA levels sufficient to confer protection of mice against severe disease progression following toxin challenge. Furthermore, we employed an mRNA-LNP encoded effector antibody, a molecular tool designed to specifically bind an epitopic tag linked to the VNAs, to prolong VNA serum half-life. Co-administration of VNA-encoding mRNA-LNPs and an effector antibody, either provided as recombinant protein or encoded by mRNA-LNP, increased serum VNA half-life in mice and in gnotobiotic piglets. Prolonged serum half-life was associated with higher concentrations of serum VNA and enhanced prophylactic protection of mice in challenge models.
Subject(s)
Clostridioides difficile , Single-Domain Antibodies , Swine , Animals , Mice , RNA , Half-Life , Antibodies , RNA, MessengerABSTRACT
Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency in functional phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine (Phe) in patients' blood and organs. Affected patients encounter severe developmental delay, neurological deficits, and behavioral abnormalities when not treated. Early diagnosis and treatment are extremely important; newborn screening programs have been implemented in most countries to ensure early identification of patients with PKU. Despite available treatment options, several challenges remain: life-long adherence to a strict diet, approval of some medications for adults only, and lack of response to these therapies in a subpopulation of patients. Therefore, there is an urgent need for treatment alternatives. An mRNA-based approach tested in PKU mice showed a fast reduction in the accumulation of Phe in serum, liver and brain, the most significant organ affected. Repeated injections of LNP-formulated mouse PAH mRNA rescued PKU mice from the disease phenotype for a prolonged period of time. An mRNA-based approach could improve the quality of life tremendously in PKU patients of all ages by replacing standard-of-care treatments.
ABSTRACT
Hereditary tyrosinemia type 1 is an inborn error of amino acid metabolism characterized by deficiency of fumarylacetoacetate hydrolase (FAH). Only limited treatment options (e.g., oral nitisinone) are available. Patients must adhere to a strict diet and face a life-long risk of complications, including liver cancer and progressive neurocognitive decline. There is a tremendous need for innovative therapies that standardize metabolite levels and promise normal development. Here, we describe an mRNA-based therapeutic approach that rescues Fah-deficient mice, a well-established tyrosinemia model. Repeated intravenous or intramuscular administration of lipid nanoparticle-formulated human FAH mRNA resulted in FAH protein synthesis in deficient mouse livers, stabilized body weight, normalized pathologic increases in metabolites after nitisinone withdrawal, and prevented early death. Dose reduction and extended injection intervals proved therapeutically effective. These results provide proof of concept for an mRNA-based therapeutic approach to treating hereditary tyrosinemia type 1 that is superior to the standard of care.
ABSTRACT
The gut microbiome has a recognized role in Non-alcoholic fatty liver disease (NAFLD) and associated comorbidities such as Type-2 diabetes and obesity. Stool transplantation has been shown to improve disease by restoring endothelial function and insulin signaling. However, more patient-friendly treatments are required. The present study aimed to test the effect of a defined bacterial consortium of nine gut commensal strains in two in vivo rodent models of Non-alcoholic steatohepatitis (NASH): a rat model of NASH and portal hypertension (PHT), and the Stelic animal (mouse) model (STAM™). In both studies the consortium was administered orally q.d. after disease induction. In the NASH rats, the consortium was administered for 2 weeks and compared to stool transplant. In the STAM™ study administration was performed for 4 weeks, and the effects compared to vehicle or Telmisartan at the stage of NASH/early fibrosis. A second group of animals was followed for another 3 weeks to assess later-stage fibrosis. In the NASH rats, an improvement in PHT and endothelial function was observed. Gut microbial compositional changes also revealed that the consortium achieved a more defined and richer replacement of the gut microbiome than stool transplantation. Moreover, liver transcriptomics suggested a beneficial modulation of pro-fibrogenic pathways. An improvement in liver fibrosis was then confirmed in the STAM™ study. In this study, the bacterial consortium improved the NAFLD activity score, consistent with a decrease in steatosis and ballooning. Serum cytokeratin-18 levels were also reduced. Therefore, administration of a specific bacterial consortium of defined composition can ameliorate NASH, PHT, and fibrosis, and delay disease progression.
ABSTRACT
BACKGROUND & AIMS: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. METHODS: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. RESULTS: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. CONCLUSION: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. LAY SUMMARY: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.
Subject(s)
Hepatocyte Nuclear Factor 4/pharmacology , Liver Cirrhosis/drug therapy , Animals , Disease Models, Animal , Hepatocyte Nuclear Factor 4/therapeutic use , Mice , RNA, Messenger/pharmacology , RNA, Messenger/therapeutic useABSTRACT
Hereditary spastic paraplegia type 5 is a neurodegenerative disease caused by loss-of-function mutations in the CYP7B1 gene encoding the oxysterol 7-α-hydroxylase involved in bile acid synthesis in the liver. Lack of CYP7B1 leads to an accumulation of its oxysterol substrates, in particular 25-hydroxycholesterol and 27-hydroxycholesterol that are able to cross the blood-brain barrier and have neurotoxic properties. A potential therapeutic strategy for SPG5 is the replacement of CYP7B1 by administration of mRNA. Here, we studied the intravenous application of formulated mouse and human CYP7B1 mRNA in mice lacking the endogenous Cyp7b1 gene. A single-dose injection of either mouse or human CYP7B1 mRNA led to a pronounced degradation of oxysterols in liver and serum within 2 days of treatment. Pharmacokinetics indicate a single injection of human CYP7B1 mRNA to be effective in reducing oxysterols for at least 5 days. Repetitive applications of mRNA were safe for at least 17 days and resulted in a significant reduction of neurotoxic oxysterols not only in liver and serum but also to some extent in the brain. Our study highlights the potential to use mRNA as a novel therapy to treat patients with SPG5 disease.
ABSTRACT
The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.
Subject(s)
Botulinum Antitoxin/immunology , Botulism/prevention & control , Immunization, Passive/methods , Post-Exposure Prophylaxis , RNA, Messenger/administration & dosage , Rabies Vaccines/immunology , Rabies/prevention & control , Animals , Antibodies, Viral/blood , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Botulinum Antitoxin/administration & dosage , Botulinum Antitoxin/blood , Botulism/therapy , Dose-Response Relationship, Immunologic , Female , Humans , Mice , Mice, Inbred BALB C , Nanoparticles , RNA, Messenger/genetics , RNA, Messenger/immunology , Rabies/therapy , Rabies Vaccines/administration & dosage , Rabies Vaccines/blood , Rabies virus/immunologyABSTRACT
BACKGROUND: Prophylactic and therapeutic vaccines often depend upon a strong activation of the innate immune system to drive a potent adaptive immune response, often mediated by a strong adjuvant. For a number of adjuvants immunological readouts may not be consistent across species. METHODS: In this study, we evaluated the innate immunostimulatory potential of mRNA vaccines in both humans and mice, using a novel mRNA-based vaccine encoding influenza A hemagglutinin of the pandemic strain H1N1pdm09 as a model. This evaluation was performed using an in vitro model of human innate immunity and in vivo in mice after intradermal injection. RESULTS: Results suggest that immunostimulation from the mRNA vaccine in humans is similar to that in mice and acts through cellular RNA sensors, with genes for RLRs [ddx58 (RIG-1) and ifih1 (MDA-5)], TLRs (tlr3, tlr7, and tlr8-human only), and CLRs (clec4gp1, clec2d, cledl1) all significantly up-regulated by the mRNA vaccine. The up-regulation of TLR8 and TLR7 points to the involvement of both mDCs and pDCs in the response to the mRNA vaccine in humans. In both humans and mice activation of these pathways drove maturation and activation of immune cells as well as production of cytokines and chemokines known to attract and activate key players of the innate and adaptive immune system. CONCLUSION: This translational approach not only allowed for identification of the basic mechanisms of self-adjuvantation from the mRNA vaccine but also for comparison of the response across species, a response that appears relatively conserved or at least convergent between the in vitro human and in vivo mouse models.
Subject(s)
Adjuvants, Immunologic/pharmacology , Genetic Engineering , Immunity, Innate/drug effects , Influenza Vaccines/immunology , RNA, Messenger/administration & dosage , Translational Research, Biomedical , Animals , Base Sequence , Dose-Response Relationship, Immunologic , Gene Expression Regulation/drug effects , Gene Regulatory Networks/drug effects , Humans , Immunity, Innate/genetics , Lymph Nodes/metabolism , Mice, Inbred C57BLABSTRACT
BACKGROUND AND OBJECTIVE: Synthetic TLR7 agonists have been proposed as oral replacements for interferonα (IFNα) therapy in the treatment of hepatitis C virus infection. However, adverse effects, such as lymphopenia and cardiovascular irregularities, have been observed in the clinical following treatment with TLR7 agonists. We wished to understand and characterise the relationship between TLR7 agonism and adverse effects. METHODS: We compared responses to two prototypic TLR7 agonists (Resiquimod: R-848; and PF-04878691) in a mouse model and compared the responses to treatment with IFNα. We measured clinically relevant adverse effects such as lymphopenia and cardiovascular irregularities and related them to plasma drug levels and clinically relevant efficacy biomarkers such as the pro-inflammatory cytokine IP-10, 2'5'OAS and TLR7 receptor expression. RESULTS: By 2 h post dose all agents had induced a dose-dependent transient lymphopenia. IFNα increased heart rate immediately following dosing, persisting for 5 h, whilst PF-04878691 induced significant reductions in blood pressure. Lymphopenia co-incided with maximum plasma drug levels, raised levels of IP-10 and the auto-induction of TLR7 expression in the blood and lymph nodes. Peak levels of 2'5'OAS occurred at 24 h post-dose and only at doses which also induced lymphopenia. CONCLUSIONS: We conclude that systemic delivery of TLR7 agonists or IFNα induces similar exaggerated pharmacology, consistent with there being a narrow therapeutic window between efficacy and safety. This clinically validated mouse model will help to investigate whether more potent agonists or optimised dosing schedules, will be successful strategies for targeting TLR7 in patients.
Subject(s)
Aminoquinolines/adverse effects , Hypotension/chemically induced , Imidazoles/adverse effects , Lymphopenia/chemically induced , Sulfonamides/adverse effects , Toll-Like Receptor 7/agonists , 2',5'-Oligoadenylate Synthetase/metabolism , Aminoquinolines/blood , Aminoquinolines/pharmacokinetics , Animals , Biomarkers/metabolism , Blood Pressure/drug effects , Chemokine CXCL10/metabolism , Female , Heart Rate/drug effects , Hypotension/metabolism , Imidazoles/blood , Imidazoles/pharmacokinetics , Interferon-alpha/adverse effects , Interferon-alpha/blood , Interferon-alpha/pharmacokinetics , Liver/drug effects , Liver/metabolism , Lymph Nodes/drug effects , Lymph Nodes/metabolism , Lymphocyte Count , Lymphopenia/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Quinolines , Sulfonamides/blood , Sulfonamides/pharmacokinetics , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolismABSTRACT
BACKGROUND: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, PF-04849285, and compares it with IFN-α2 and pegylated IFN-α2; the latter being the current standard of care for HCV. METHODS: The antiviral properties were evaluated in vitro using the HCV replication assay (replicon) and the general encephalomyocarditis virus assay. The binding affinity to both IFNR-subunits was assessed using surface plasmon resonance. Ex vivo experiments using cynomolgus monkey and human blood were used for the evaluation of induction of IFN-inducible biomarkers (interferon inducible protein 10 [IP-10], 2'-5'-oligoadenylate synthetase [OAS2] and interleukin-6 [IL-6]). The molecule was tested intravenously and subcutaneously in cynomolgus monkey in a single dose study for two weeks at 0.01, 1, 5 and 20 mg/kg. Each route and dose combination was given to a single male animal, blood samples were collected for evaluation of biomarkers and pharmacokinetics. The compound was also tested in cynomolgus monkey in a multiple dose study for four weeks, with a twice-a-week dosing prior to a three-week wash-out period for toxicokinetics, pharmacokinetics, and biomarker evaluation at 20, 50 or 100 mg/kg subcutaneously and 20 mg/kg intravenously. RESULTS: The molecule is 10× more potent than the pegylated IFN-α2a, with potency similar to the unmodified IFN-α2a. No unanticipated findings were observed in cynomolgus monkey when dosed up to 20 mg/kg, >10,000-fold margin over the anticipated efficacious human dose. CONCLUSIONS: The biomarker and toxicological findings were consistent with a potent IFN molecule. The potency and pharmacokinetic properties of the molecule are consistent with dosing at least every two weeks with the potential for monthly dosing' and not 'at least twice daily' as presented in the original [corrected].
Subject(s)
Antiviral Agents/pharmacology , Hepatitis C/drug therapy , Interferon-alpha/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/toxicity , Cell Line , Drug Evaluation, Preclinical , Encephalomyocarditis virus/drug effects , Female , Hepacivirus/drug effects , Humans , Interferon alpha-2 , Interferon-alpha/administration & dosage , Interferon-alpha/pharmacokinetics , Interferon-alpha/toxicity , Macaca fascicularis , Male , Receptors, Interferon/metabolism , Recombinant Fusion Proteins/pharmacokinetics , Recombinant Fusion Proteins/toxicity , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Treatment Outcome , Virus Replication/drug effectsABSTRACT
In the past, antiviral research has focused mainly on viral targets. As the search for effective and differentiated antiviral therapies continues, cellular targets are becoming more common, bringing with them a variety of challenges and concerns. Toll-like receptors (TLRs) provide a unique mechanism to induce an antiviral state in the host. In this review we introduce TLRs as targets for the pharmaceutical industry, including how they signal and thereby induce an antiviral state through the production of type I interferons. We examine how TLRs are being therapeutically targeted and discuss several clinically precedented agents for which efficacy and safety data are available. We describe some of the chemistries that have been applied to both small molecule and large molecule leads to tune agonist potency, and offer a differentiated safety profile through targeting certain compartments such as the gut or the lung, thereby limiting systemic drug exposure and affecting systemic cytokine levels. The application of low-dose agonists of TLRs as vaccine adjuvants or immunoprotective agents is also presented. Some of the challenges presented by this approach are then discussed, including viral evasion strategies and mechanism-linked inflammatory cytokine induction.
Subject(s)
Antiviral Agents/administration & dosage , Immunologic Factors/administration & dosage , Toll-Like Receptors/agonists , Virus Diseases/drug therapy , Virus Diseases/immunology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Humans , Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Interferons/immunology , Interferons/metabolismABSTRACT
The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepatitis C/drug therapy , Interferons/agonists , Toll-Like Receptor 7/agonists , Aldehyde Oxidase/metabolism , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Dose-Response Relationship, Drug , Drug Discovery , Drug Evaluation, Preclinical , Hepacivirus/physiology , Hepatitis C/virology , Humans , Injections, Intravenous , Interferon Inducers/chemical synthesis , Interferon Inducers/chemistry , Interferon Inducers/pharmacokinetics , Interferon Inducers/pharmacology , Microsomes, Liver/metabolism , Molecular Targeted Therapy , Molecular Weight , Purines/chemical synthesis , Purines/metabolism , Rats , Solubility , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2',5'-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by â¼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by â¼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.
Subject(s)
Antiviral Agents/therapeutic use , Endopeptidases/genetics , Interferon-alpha/therapeutic use , Ribavirin/therapeutic use , Blotting, Western , Cell Line , Humans , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics , Ubiquitin ThiolesteraseABSTRACT
The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.
Subject(s)
Antiviral Agents/chemistry , Hepacivirus/drug effects , Purines/chemistry , RNA Virus Infections/drug therapy , Toll-Like Receptor 7/agonists , Administration, Oral , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/therapeutic use , Drug Design , Mice , Models, Animal , Purines/pharmacokinetics , Purines/therapeutic use , Rats , Structure-Activity Relationship , Toll-Like Receptor 7/metabolismABSTRACT
Toll-like receptors recognize invading microorganisms and activate innate immune responses. Their discovery has opened up a range of therapeutic possibilities, in particular for infectious diseases. Responses to TLR agonists have been largely studied in mice and little information exists in other species. Given that rats are commonly used for pharmacokinetic and toxicology studies in drug development, we compared TLR7 responses in rat and human. Stimulation of rat and human peripheral blood mononuclear cells with the TLR7 agonist SM360320 showed that in rat cells, the interferon-induced gene, 2', 5' oligoadenylate synthase and tumor necrosis factor alpha were induced at lower concentrations and to a greater degree compared with human cells. Both human and rat cells demonstrated tolerance and could not be restimulated following initial treatment with high concentrations of SM360320. Reducing the concentration of the initial treatment allowed cells to be restimulated following a period of recovery. The initial treatment concentration had to be reduced to a greater extent to enable restimulation of rat cells compared with human cells. Dosing whole rats repeatedly with different concentrations of SM360320 confirmed the in vitro results. Treatment of human cells with high concentrations of interferon alpha did not induce tolerance to subsequent treatment with SM360320 indicating that tolerance occurs in the TLR7 signaling pathway, rather than the interferon signaling pathway. We conclude that rat and human cells respond differently to TLR7 activation and that these differences should be considered when using rat as a model to study TLR7 agonists.
Subject(s)
Leukocytes, Mononuclear/immunology , Toll-Like Receptor 7/agonists , Tumor Necrosis Factor-alpha/metabolism , 2',5'-Oligoadenylate Synthetase/drug effects , 2',5'-Oligoadenylate Synthetase/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Humans , Interferon-alpha/pharmacology , Leukocytes, Mononuclear/drug effects , Rats , Signal Transduction/drug effects , Signal Transduction/immunology , Toll-Like Receptor 7/immunology , Tumor Necrosis Factor-alpha/agonistsABSTRACT
Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2',5'-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Toll-Like Receptors/agonists , Cell Line, Tumor , Cytokines/metabolism , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Toll-Like Receptor 3/agonists , Toll-Like Receptor 7/agonists , Toll-Like Receptor 9/agonistsABSTRACT
Discovery of potential therapeutics against hepatitis C virus (HCV) infection has been hampered in the past decade by the inability to grow this virus in tissue culture and by the lack of robust small animal models. This situation has been improved by the recent development of a selectable HCV replicon cell culture system. For the first time, drug discovery scientists are able to screen large compound collections using the replicon cell culture system to identify small molecules with the potential to inhibit HCV RNA replication. The replicon system has also been used to elucidate inhibitors' antiviral mechanism of action and to optimize antiviral potency. In this review, we will summarize the recent development of HCV replicon cell culture system and its use in anti-HCV drug discovery. The antiviral activities of promising lead compounds are also reviewed.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/physiology , Hepatitis C/drug therapy , Replicon/physiology , Virus Replication/physiology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Hepacivirus/enzymology , Hepatitis C/virology , Humans , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Tumor Cells, Cultured , Virus Cultivation/methods , Virus Replication/drug effectsABSTRACT
We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.
Subject(s)
DNA-Binding Proteins/physiology , Diarrhea Viruses, Bovine Viral/genetics , Replicon , Transcription Factors/physiology , Animals , Antiviral Agents/pharmacology , Cattle , Cell Line , Diarrhea Viruses, Bovine Viral/drug effects , Diarrhea Viruses, Bovine Viral/physiology , Genome, Viral , Hepacivirus/drug effects , Hepacivirus/genetics , Hepacivirus/physiology , Humans , Interferon Regulatory Factor-3 , Interferons/pharmacology , Mutation , RNA, Viral/biosynthesis , RNA, Viral/genetics , Sendai virus/physiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/physiology , Virus ReplicationABSTRACT
In a rat model of Borna disease, intracerebral ribavirin caused clinical improvement without changes in virus titer or nucleic acid. Levels of microglia and infiltrating CD4 and CD8 cells were decreased, despite increases in mRNAs encoding interleukin-1beta (IL-1beta), IL-10, and gamma interferon in the brain. Intracerebral ribavirin may reduce morbidity through effects on microglia cell proliferation.