A novel antiviral strategy targeting human metapneumovirus through pH modulation in human airway epithelial cells

Human metapneumovirus (hMPV) is a major cause of respiratory infections, particularly in vulnerable populations, yet no approved vaccines or targeted antivirals are available. pH-regulated processes, including airway epithelial physiology, endosomal acidification and viral fusion mediated by the fusion (F) protein, are critical for hMPV infection. This study evaluates PHOH-001, an inhaled alkaline buffer, as a potential therapeutic strategy to modulate airway pH and inhibit hMPV infection. Using rhMPV-GFP (green fluorescent protein-expressing hMPV), viral replication was assessed in primary human airway epithelial cells (HAECs). PHOH-001 significantly reduced GFP expression at 72 h post-infection in both submerged and air-liquid interface cultures, with effects comparable to those of the endosomal acidification inhibitor bafilomycin A1. In Vero E6 cells, PHOH-001 increased extracellular and intracellular pH in a dose-dependent manner and correspondingly reduced hMPV infection. In HAECs, PHOH-001 treatment reduced early virus-cell interactions, as measured by decreased cell-associated viral RNA under conditions restricting internalization. PHOH-001 also reduced viral replication, as measured by TCID₅₀ assays and decreased syncytium formation. In parallel, PHOH-001 increased endosomal pH, consistent with altered endosomal acidification. Furthermore, PHOH-001 altered F protein localization and coincided with changes in actin organization, consistent with impaired viral spread. Collectively, these findings indicate that PHOH-001 modulates multiple pH-dependent steps in hMPV infection in vitro and support airway pH modulation as a potential antiviral strategy against hMPV.