(498) CFTR Modulator Therapy Increases Viral Mobility in Primary CF Airway Cultures Infected with RSV

Background: Respiratory syncytial virus (RSV) infects the upper and lower airways in young children, causing cell shedding, mucus hypersecretion and bronchiolitis, which in CF, can produce severe disease. Individuals with CF suffer from thick mucus accumulation and impaired mucociliary clearance, resulting in high susceptibility to respiratory viruses. Highly effective CFTR modulators have greatly improved the quality of life for individuals living with CF, as these therapies reverse the aberrant mucus properties and restore airway clearance. We examined the effects of RSV infection in disease-control and CF primary human bronchial epithelial (HBE) cell cultures, as well as the effects of ETI treatment on RSV mobility.

Methods: HBE cells from non-CF donors (NCF) and CF donors with the F508del mutation (CF) were grown on ALI and treated with vehicle or ETI for 3 days prior to infection with GFP-tagged RSV. As thick mucus adheres to the CF airway surfaces, viral access to the cells was examined in the presence or absence of apical mucus. Viral mobility was quantified as the % area of GFP signal in infected cultures (e.g., viral spread). ASL height was measured via confocal microscopy. RT-qPCR was used to quantify viral, CFTR, and ENaC mRNA expression. Biophysical properties of mucus were assessed via % solids and cilia dynamics via video tracking.

Results: In RSV infected NCF cultures, ASL height significantly increased compared to mock. In contrast, ASL height failed to increase in CF cultures in response to RSV infection. RT-qPCR showed no change in CFTR mRNA expression between mock and RSV-infected NCF cells but β-ENaC and λ-ENaC expression were decreased, suggesting that ASL hydration was linked to ENaC downregulation. Analysis of GFP signal revealed a significant increase in viral mobility in NCF compared to CF cells. When NCF and CF were treated with ETI, viral mobility was significantly increased compared to the vehicle groups. ETI treatment of CF cells resulted in decreased mucus % solids and increased mucociliary transport. Removal of mucus prior to infection increased viral mobility in both NCF and CF cells, independently of ETI treatment. Increased viral mobility correlated with increased viral mRNA in NCF and CF cells treated with ETI compared to CF cells without treatment, which can be explained by the fact that in vitro systems cannot clear and eradicate particles, in contrast with the lungs. Therefore, viral mobility without annihilation of the virus increases the odds of infecting new cells. More work needs to be done to generate an open system that will permit the clearance of particles.

Conclusions: Children with CF experience more severe disease caused by RSV infection compared to the general population. This is in part due to impaired clearance of pathogens. In this study, we demonstrated that RSV infection of NCF cultures resulted in ASL hydration, which correlated with increased viral mobility. In contrast, RSV infected CF cultures failed to hydrate the ASL, which correlated with reduced viral mobility. ETI treatment restored viral mobility in CF cultures and further increased viral mobility in NCF cultures, suggesting that CFTR modulators may have positive effects on RSV infection for CF and also for NCF individuals. We anticipate that increased viral mobility will correlate with decreased viral load in vivo, as suggested by increased MCT and lower mucus % solids following ETI treatment as shown in vitro.

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