(550) A safe inhaled alkaline medication that alters airway pH in cystic fibrosis and inhibits RSV respiratory infection

Background: Although outcomes for people with cystic fibrosis (CF) have improved over the past decade with the advent of CFTR modulators, progressive decline in lung function and eventual respiratory failure is still the major cause of morbidity and mortality. Pulmonary exacerbations (PEs) play an important role in worsening pulmonary function in CF, and respiratory viruses are a leading trigger of PEs. Respiratory syncytial virus (RSV) is one of the most common viruses isolated in children with CF during a PE. Loss of CFTR function leads to decreased secretion of HCO3-. This reduction in HCO3- secretion contributes to an abnormally acidic extracellular pH which decreases mucociliary clearance, impairs host defense, and increases viral loads within the CF airways. It likely also contributes to the more alkaline intracellular pH seen in CF human airway epithelial cells (HAECs). Of note, most respiratory viruses require an acidic intracellular pH, specifically acidic endosomal pH, to infect HAECs and replicate. Therefore, the more alkaline intracellular pH found in CF may be relatively protective against some viral infections. Inhaled Optate (Airbase BC, Indianapolis) safely raises airway surface and intracellular pH. We plan to study the effects of further increasing intracellular pH on RSV infection in CF and non-CF HAECs. We hypothesize that the relatively alkaline intracellular pH in CF HAECs is protective with respect to RSV infection compared to non-CF HAECs, and administration of the base, Optate, will further decrease RSV infection in these cells.

Methods: HAECs from a healthy subject were cultured at both at air-liquid interface (ALI) and submerged. HAECs were infected with green fluorescent protein-labeled RSV (GFP-RSV; multiplicity of infection of 1) and treated with Optate or with phosphate buffered saline (PBS) control. Supernatant was collected after a 4-hour incubation period and then every 24 hours for 3 or 10 days. We used fluorescence intensity, fluorescent particle counts, and plaque assays to quantitate GFP-RSV infection.

Results: In submerged culture, RSV infection was lower in cells treated with Optate compared to PBS control at 48 and 72 hours: there was both a dose-dependent decrease in fluorescent intensity during infection (48H p=0.0174, 72H p=< 0.0001), and a decrease in fluorescent particle count (48H p=0.0178, 72H p=0.0019) and in plaque forming units (PFUs) (48H p=0.0011, 72H p=0.0148) from supernatant. Immediately following incubation period, supernatant collected from Optate treated cells had higher fluorescent particle count (p=0.0187) and PFUs (p=0.0064) compared to control. RSV infection was also decreased in cells treated with Optate at ALI as noted by a decrease in fluorescence intensity from 144 to 240 hours during primary infection (p £ 0.01).

Conclusions: Optate, an inhaled anti-inflammatory agent being studied in CF, inhibits airway epithelial RSV infection. These findings suggest that Optate could be beneficial in RSV-associated PEs. In addition, its mechanism of action (airway pH modulation) is mutation agnostic and could be administered to all people with CF.

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