Development of in vitro airway epithelial model to assess immune response and safety of mucosal adjuvants

Mucosal vaccines delivered intranasally or by inhalation, can elicit localized immunity that protects mucosal tissues at the site of infection and may reduce transmission of respiratory viruses, including respiratory syncytial virus (RSV), influenza virus, and SARS-CoV-2. Many mucosal vaccine candidates incorporate adjuvants to enhance and broaden immune response, underscoring the need for safe and effective formulations targeting the respiratory mucosa. Here, we employed differentiated human nasal and bronchial epithelial cells (hNEC and hBEC) cultured at air-liquid interface (ALI) to evaluate the soybean oil-in-water intranasal NE01 adjuvant, recently assessed clinically. Kinetic and dose-response studies showed that hBECs were more sensitive than hNEC to the cytopathic effect of NE01 as evidenced by loss of barrier integrity, reduced viability, and Interleukin-8 production. Spiking Cetylpyridinium chloride (CPC), a component of NE01, into a squalene oil-in-water adjuvant identified CPC as a potential contributor to the observed cytotoxicity in hBEC. Lower NE01 concentrations did not compromise hBEC viability or barrier function, indicating concentration-dependent effects and establishing thresholds for epithelial perturbation. The data provide proof-of-concept supporting the ALI system for assessing the effects of mucosal adjuvants on Upper and Lower respiratory tract epithelia during the early stages of development. This approach may help prioritize candidates while reducing reliance on animal use in preclinical evaluation.