RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy (original) (raw)
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Antiviral Research, 2008
Small interfering RNAs (siRNAs) work through RNA interference (RNAi), the natural RNA inhibitory pathway, to down-regulate protein production by inhibiting targeted mRNA in a sequence-specific manner. ALN-RSV01 is an siRNA directed against the mRNA encoding the N-protein of respiratory syncytial virus (RSV) that exhibits specific in vitro and in vivo anti-RSV activity. The results of two safety and tolerability studies with ALN-RSV01 involving 101 healthy adults (65 active, 36 placebo, single-and multiple dose, observer-blind, randomized doseescalation) are described. Intranasal administration of ALN-RSV01 was well tolerated over a dose range up through 150 mg as a single dose and for five daily doses. Adverse events were similar in frequency and severity to placebo (normal saline) and were transient, mild to moderate, with no dose-dependent trend. The frequency or severity of adverse events did not increase with increasing ALN-RSV01 exposure. All subjects completed all treatments and assessments with no early withdrawals or serious adverse events. Physical examinations, vital signs, ECGs and laboratory tests were normal. Systemic bioavailability of ALN-RSV01 was minimal. ALN-RSV01 appears safe and well tolerated when delivered intranasally and is a promising therapeutic candidate for further clinical development.
Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was cotransfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6 days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection. .my (S. Zamberi). . The G protein is responsible for the attachment of the virus to the cell, while the F protein is involved in viral penetration, fusion and syncytium formation. The small hydrophobic (SH), matrix (M) and M2 proteins are virus transcription factors, while the nucleoprotein (N), phosphoprotein (P) and the large (L) protein are RNA dependent RNA polymerases present in the nucleocapsid of the RSV. The small hydrophobic proteins have been shown to be effectively inhibited in previous study . The non-structural proteins (NS1 and NS2) are only found in infected cells . RSV M2-2 protein (90 amino acids for strain A2) is a small protein encoded by a second, downstream ORF in the M2 mRNA that slightly overlaps the 5 -proximal M2-1 ORF . M2-2 protein provides an initial http://dx.
Differential antiviral activities of RSV inhibitors in human airway epithelium
ABSTRACTWe report the use of reconstituted 3D-human airway epithelium cells of bronchial origin (HuAEC) in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. RSV-A replicates efficiently in HuAEC and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleosides) elicited a marked antiviral effect even when start of treatment was delayed until one or even three days after infection. Levels of the inflammation marker RANTES (mRNA) increased ∼200-fold in infected-untreated cultures (at three weeks post infection), but levels were comparable to those of uninfect...
Antimicrobial Agents and Chemotherapy
Effective treatments for respiratory syncytial virus (RSV) infection are lacking. We report a human proof-of-concept study for RV521, a small-molecule antiviral inhibitor of the RSV-F protein. In this randomized, double-blind, placebo-controlled trial (NCT03258502), healthy adults were challenged with RSV-A Memphis-37b. After infection was confirmed (or 5 days after challenge virus inoculation), subjects received RV521 (350 mg or 200 mg) or placebo, orally every 12 hours for 5 days. Primary endpoint was area under the curve (AUC) for viral load, as assessed by reverse transcriptase quantitative PCR (RT-qPCR), in nasal wash samples. Primary efficacy analysis set included subjects successfully infected with RSV who received ≥1 dose of study drug. Sixty-six subjects were enrolled (n=22 per group); 53 were included in the primary analysis set (RV521 350 mg: n=16; 200 mg: n=18; placebo: n=19). Mean AUC of RT-qPCR-assessed RSV viral load (log10 PFUe/mL x hours) was significantly lower wit...
The Journal of antimicrobial chemotherapy, 2018
We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. HuAECs were infected with RSV-A Long strain (0.01 CCID50/cell, where CCID50 represents 50% cell culture infectious dose in HEp2 cells) on the apical compartment of the culture. At the time of infection or at 1 or 3 days post-infection, selected inhibitors were added and refreshed daily on the basal compartment of the culture. Viral shedding was followed up by apical washes collected daily and quantifying viral RNA by RT-qPCR. RSV-A replicates efficiently in HuAECs and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post-infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only...
Journal of Biological Chemistry, 2018
Edited by George N. DeMartino Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hitto-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratoryadapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.
Challenges and opportunities in developing respiratory syncytial virus therapeutics
The Journal of infectious diseases, 2015
Two meetings, one sponsored by the Wellcome Trust in 2012 and the other by the Global Virology Foundation in 2013, assembled academic, public health and pharmaceutical industry experts to assess the challenges and opportunities for developing antivirals for the treatment of respiratory syncytial virus (RSV) infections. The practicalities of clinical trials and establishing reliable outcome measures in different target groups were discussed in the context of the regulatory pathways that could accelerate the translation of promising compounds into licensed agents. RSV drug development is hampered by the perceptions of a relatively small and fragmented market that may discourage major pharmaceutical company investment. Conversely, the public health need is far too large for RSV to be designated an orphan or neglected disease. Recent advances in understanding RSV epidemiology, improved point-of-care diagnostics, and identification of candidate antiviral drugs argue that the major obstac...
Antiviral efficacy of an RSV fusion inhibitor in a bovine model of RSV infection
Antimicrobial Agents and Chemotherapy, 2015
word count: 238 31 Text word count: 6248 32 ABSTRACT 33 34 Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants. 35 Effective treatment for RSV infection is a significant unmet medical need. While new RSV 36 therapeutics are now in development, there are very few animal models that mimic the 37 pathogenesis of human RSV, making it difficult to evaluate new disease interventions. 38 Experimental infection of Holstein calves with bovine RSV (bRSV) causes a severe respiratory 39 infection that is similar to human RSV infection, providing a relevant model for testing novel 40 therapeutic agents. In this model, viral load is readily detectable in nasal secretions by qRT-PCR 41 and cumulative symptom scoring together with histopathology evaluations of infected tissue 42 allow for the assessment of disease severity. The bovine RSV model was used to evaluate the 43 antiviral activity of a RSV fusion inhibitor, GS1, which blocks virus entry by inhibiting the 44 fusion of the viral envelope with the host cell membrane. The efficacy of GS1, a close structural 45 analog of GS-5806 that is being developed to treat RSV infection in humans was evaluated in 46 two randomized, blinded, placebo controlled studies in bRSV-infected calves. Intravenous 47 administration of GS1 at 4 mg/kg/day for 7 days starting at 24 h or 72 h post-inoculation 48 provided clear therapeutic benefit by reducing the viral load, disease symptom scores, respiration 49 rates and lung pathology associated with bRSV infection. These data support the use of the 50 bovine RSV model for evaluation of experimental therapeutics for treatment of RSV. 51 52 53 Respiratory syncytial virus (RSV) is a member of the Paramyxoviridae family. RSV is an 54 enveloped virus with a negative single-strand ribonucleic acid (RNA) genome that encodes for 55 11 proteins, including 3 surface glycoproteins (F, G, and SH) and 4 proteins that comprise the 56 viral RNA polymerase complex (N, P, L, and M2-1). The virus replicates in the ciliated 57 respiratory epithelial cells that line the respiratory tract. Replication of the virus can induce 58 syncytia (cell fusion) that can be readily observed in transformed cell lines and has also been 59 detected in infant lung tissue from fatal cases of RSV infection (1). However, replication of RSV 60 in primary human airway epithelial cells does not cause measurable cytopathic effects (2). While 61 direct virus-induced cytopathic effects may not be the primary cause of clinical disease, virus 62 replication activates the host immune responses which can lead to immune-mediated 63 pathogenesis (3). 64 65 RSV circulates in the population as a single serotype with two major antigenic subgroups, A and 66 B, that cause seasonal infections during fall and winter in the US and other temperate regions of 67 the world (4). Children younger than 2 years of age, immunocompromised individuals, and 68 adults with underlying respiratory dysfunction such as asthma or COPD are at greater risk for 69 developing complications due to RSV (5-10). While most infections resolve without medical 70 intervention, RSV infection can cause acute bronchiolitis and pneumonia in infants and in elderly 71 adults (5, 11). Infection of the lower respiratory tract can cause severe pneumonia requiring 72 hospitalization and resulting in mortality. 73 74 Despite extensive study of the clinical features and immunopathogenesis of RSV, there are no 75 effective therapeutics or vaccines to treat RSV infection. A prophylactic monoclonal antibody 76 Synagis ® (palivizumab) is recommended for preventing RSV infection among infants at high risk 77 for lower respiratory tract RSV infection (12). Vaccine strategies using formalin-fixed RSV as 78 the immunogen did not fully protect infants from RSV infection and in some cases enhanced 79 disease, underscoring the challenges of developing effective infant vaccines for RSV (13). The 80 only approved antiviral treatment for RSV is Virazole ® (ribavirin), which is delivered as an 81 inhaled agent. Virazole has shown equivocal efficacy in bone marrow transplant patients and is 82 4 not typically used to treat infants with RSV due to poor efficacy and tolerability, as well as the 83 complexity of the specialized aerosol drug delivery system (14-16). A number of small molecule 84 inhibitors of RSV have been evaluated as potential therapeutics for treating RSV infections that 85 target viral entry and replication (17-23). While several clinical candidates have been evaluated 86 in human studies, only ribavirin has been approved for the treatment of RSV infection. 87 88 Preclinical development of new therapeutics requires evaluation of efficacy in animal models of 89 RSV infection. The cotton rat model is commonly used to measure RSV replication and evaluate 90 RSV therapeutics (24, 25). While cotton rats are permissive to RSV infection, virus replication 91 does not produce quantifiable symptoms and the extent of virus infection is measured by 92 determining viral lung titers from infected animals following euthanasia. Thus, the cotton rat 93 model is not suitable for measuring changes in disease pathogenesis resulting from antiviral 94 therapy. Additional animal models have been developed using chimpanzees, African green 95 monkeys, infant Rhesus monkeys, and mice to study RSV infection (18, 26-29). As in the cotton 96 rat model, virus replication does not induce significant disease and symptoms tend to be mild or 97 absent. Infection of neonatal lambs with RSV produces mild symptoms that include fever, 98 tachypnea, and malaise as well as histologic lesions in lung tissue (27, 30). However, the natural 99 history of RSV infection in this model has not been fully characterized and there is limited data 100 on quantitative viral load measurements in tissues over the course of infection or on the 101 development of techniques to quantify symptoms to correlate virus replication with disease 102 progression. 103 104 Bovine RSV (bRSV) is a natural pathogen of cattle that causes a disease in young animals 105 similar to hRSV in humans (31, 32). The virus replicates in the upper and lower respiratory tract 106 to produce a spectrum of illness ranging from subclinical to severe bronchiolitis and pneumonia 107 (33). The similarity in pathogenesis between bRSV and hRSV is underscored by studies that 108 have demonstrated disease enhancement by a formalin-inactivated bRSV/alum vaccine made 109 using the protocol that created the disease enhancing RSV vaccine the sickened and killed 110 several children in the 1960's (13, 34). 111 112 5 The goal of this study was to develop a preclinical animal model to evaluate efficacy of 113 experimental therapeutics for treatment of human RSV infection. To validate the bRSV model 114 for testing antiviral therapeutic interventions, we tested the therapeutic efficacy of a novel RSV 115 fusion inhibitor GS1, a close structural analog of GS-5806 which has recently been shown to 116 reduce RSV viral load and clinical symptoms in a human model of experimental RSV infection 117 (Figure 1) (20). The effects of GS1 on bRSV replication and disease symptoms were assessed in 118 two randomized, placebo controlled, blinded efficacy studies in calves less than 8 weeks old to 119 assess GS1 efficacy. The results demonstrate that the bRSV infection of young calves is a 120 suitable model for quantifying the impact of antiviral intervention on the viral load and disease 121 symptoms and can be used to evaluate the in vivo efficacy of RSV antiviral compounds. 122 6 MATERIALS AND METHODS 123 124 Compounds 125 GS1 was synthesized by the Department of Medicinal Chemistry, Gilead Sciences (Foster City, 126 CA) (Figure 1). 127 128 Cells and Viruses 129 Primary bovine turbinate (BT) cells were grown in Dulbecco's modified Eagle's medium 130 (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/mL), and 131 streptomycin (100 μg/mL) at 37°C with 5% CO 2 . Primary bovine alveolar type 2 (BAT2) cells 132 were isolated from newborn calf lung as described previously (35). Cells were grown in DMEM-133 keratinocyte medium at a ratio of 1:1 (Invitrogen, Carlsbad, CA), supplemented with 2% FBS, 5 134 mM l-glutamine, 0.02% lactalbumin dehydrogenase, and penicillin (100/mL)-streptomycin (100 135 μg/mL) (Invitrogen), at 37°C in a humidified incubator with 5% CO 2 . 136 137 Stocks of bRSV were prepared from a clinical isolate of a virulent bRSV strain (CA-1) as 138 previously described (36, 37). Virus stocks were prepared by infecting BT cell monolayer in a 139 T75 culture flask with 3 × 10 5 plaque forming units (pfu) of virus. The infected cells were 140 incubated until viral cytopathic effects (CPE) reached approximately 30 to 50% of the cell 141 monolayer, usually by day 5 post infection. The cultures were harvested and subjected to a single 142 freeze thaw cycle to release cell associated virus. Aliquots of virus suspensions were placed on 143 ice and administered to calves by aerosol within approximately one hour. Additional aliquots 144 were used to quantify viral titer by plaque assay and any remaining virus was frozen at -80°C. 145 146 In Vitro Antiviral Activity 147 The antiviral activity of GS1 against bRSV was measured on bovine turbinate (BT) and bovine 148 alveolar type 2 (BAT2) cells using a cell viability assay that measures cellular ATP levels. Serial 149 3-fold dilutions of GS1 prepared in DMSO and added to cell culture media at 2X the final 150 concentration. Cell monolayers were prepared in 96-well plates at 1 x 10 4 cells per well and the 151 next day infected with bRSV at 0.05 pfu/cell (MOI 0.05) in 100 μL final volume. A parallel set 152 of plates was prepared and mock-infected to measure compound cytotoxicity. The assay plates 153 Cell Immune Response during Respiratory Virus Infection. J Virol 86:5422-5436.
Journal of medicinal chemistry, 2015
Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of…