Abstract
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.
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References
Zhang, Z. et al. Mucosal immunity and vaccination strategies: current insights and future perspectives. Mol. Biomed. 6, 57 (2025).
Jung, J. et al. Transmission and infectious SARS-CoV-2 shedding kinetics in vaccinated and unvaccinated individuals. JAMA Netw. Open 5, e2213606 (2022).
Puhach, O. et al. Infectious viral load in unvaccinated and vaccinated individuals infected with ancestral, Delta or Omicron SARS-CoV-2. Nat. Med. 28, 1491–1500 (2022).
Singanayagam, A. et al. Community transmission and viral load kinetics of the SARS-CoV-2 delta (B.1.617.2) variant in vaccinated and unvaccinated individuals in the UK: a prospective, longitudinal, cohort study. Lancet Infect. Dis. 22, 183–195 (2022).
Lavelle, E. C. & Ward, R. W. Mucosal vaccines - fortifying the frontiers. Nat. Rev. Immunol. 22, 236–250 (2022).
Macpherson, A. J., McCoy, K. D., Johansen, F. E. & Brandtzaeg, P. The immune geography of IgA induction and function. Mucosal Immunol. 1, 11–22 (2008).
Sinha, D., Yaugel-Novoa, M., Waeckel, L., Paul, S. & Longet, S. Unmasking the potential of secretory IgA and its pivotal role in protection from respiratory viruses. Antivir. Res. 223, 105823 (2024).
Mao, T. et al. Unadjuvanted intranasal spike vaccine elicits protective mucosal immunity against sarbecoviruses. Science 378, eabo2523 (2022).
Boyaka, P. N. Inducing mucosal IgA: A challenge for vaccine adjuvants and delivery systems. J. Immunol. 199, 9–16 (2017).
Li, J. et al. Advances and prospects of respiratory mucosal vaccines: mechanisms, technologies, and clinical applications. NPJ Vaccines 10, 230 (2025).
Zeng, L. Mucosal adjuvants: opportunities and challenges. Hum. Vaccin. Immunother. 12, 2456–2458 (2016).
Maciel, M. Jr. et al. Evaluation of the reactogenicity, adjuvanticity and antigenicity of LT(R192G) and LT(R192G/L211A) by intradermal immunization in mice. PLoS ONE 14, e0224073 (2019).
Seok, J. et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. USA 110, 3507–3512 (2013).
Zeiss, C. J. & Brayton, C. F. Immune responses to the real world. Lab Anim. 47, 13–14 (2018).
Grainger, C. I., Greenwell, L. L., Lockley, D. J., Martin, G. P. & Forbes, B. Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier. Pharm. Res. 23, 1482–1490 (2006).
Silva, S., Bicker, J., Falcão, A. & Fortuna, A. Air-liquid interface (ALI) impact on different respiratory cell cultures. Eur. J. Pharm. Biopharm. 184, 62–82 (2023).
Kesimer, M. et al. Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, L92–L100 (2009).
Prytherch, Z. et al. Tissue-specific stem cell differentiation in an in vitro airway model. Macromol. Biosci. 11, 1467–1477 (2011).
Fulcher, M. L. & Randell, S. H. Human nasal and tracheo-bronchial respiratory epithelial cell culture. Methods Mol. Biol. 945, 109–121 (2013).
Rayner, R. E., Makena, P., Prasad, G. L. & Cormet-Boyaka, E. Optimization of normal human bronchial epithelial (NHBE) cell 3D cultures for in vitro lung model studies. Sci. Rep. 9, 500 (2019).
Zhao, C. et al. Activation of STAT3-mediated ciliated cell survival protects against severe infection by respiratory syncytial virus. J. Clin. Invest. 134, e183978 (2024).
Hou, Y. J. et al. SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract. Cell 182, 429–446 e414 (2020).
Liang, K. et al. Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium. Sci. Immunol. 9, eadn0178 (2024).
Stolting, H. et al. Distinct airway epithelial immune responses after infection with SARS-CoV-2 compared to H1N1. Mucosal Immunol. 15, 952–963 (2022).
Major, J. et al. Type I and III interferons disrupt lung epithelial repair during recovery from viral infection. Science 369, 712–717 (2020).
Song, B. M., Kang, Y. M., Kim, H. S. & Seo, S. H. Induction of inflammatory cytokines and toll-like receptors in human normal respiratory epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, and highly pathogenic H5N1 influenza virus. Viral Immunol. 24, 179–187 (2011).
Forero, A. et al. Evaluation of the innate immune responses to influenza and live-attenuated influenza vaccine infection in primary differentiated human nasal epithelial cells. Vaccine 35, 6112–6121 (2017).
Makidon, P. E. et al. Pre-clinical evaluation of a novel nanoemulsion-based hepatitis B mucosal vaccine. PLoS ONE 3, e2954 (2008).
Hamouda, T., Sutcliffe, J. A., Ciotti, S. & Baker, J. R. Jr. Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant. Clin. Vaccin. Immunol. 18, 1167–1175 (2011).
Stanberry, L. R. et al. Safety and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens. Vaccine 30, 307–316 (2012).
Das, S. C. et al. Nanoemulsion W805EC improves immune responses upon intranasal delivery of an inactivated pandemic H1N1 influenza vaccine. Vaccine 30, 6871–6877 (2012).
Wang, S. H. et al. Recombinant H5 hemagglutinin adjuvanted with nanoemulsion protects ferrets against pathogenic avian influenza virus challenge. Vaccine 37, 1591–1600 (2019).
Smith, D., Streatfield, S. J., Acosta, H., Ganesan, S. & Fattom, A. A nanoemulsion-adjuvanted intranasal H5N1 influenza vaccine protects ferrets against homologous and heterologous H5N1 lethal challenge. Vaccine 37, 6162–6170 (2019).
Deming, M. E. et al. An intranasal adjuvanted, recombinant influenza A/H5 vaccine primes against diverse H5N1 clades: a phase I trial. Nat. Commun. 16, 9321 (2025).
Roy, C. J. et al. Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method. Respir. Res. 12, 153 (2011).
Xu, H., Cai, L., Hufnagel, S. & Cui, Z. Intranasal vaccine: factors to consider in research and development. Int. J. Pharm. 609, 121180 (2021).
Jeyanathan, M. et al. Induction of lung mucosal immunity by a next-generation inhaled aerosol COVID-19 vaccine: an open-label, multi-arm phase 1 clinical trial. Nat. Commun. 16, 6000 (2025).
Huvenne, W. et al. Different regulation of cigarette smoke induced inflammation in upper versus lower airways. Respir. Res. 11, 100 (2010).
Sahakijpijarn, S., Smyth, H. D. C., Miller, D. P. & Weers, J. G. Post-inhalation cough with therapeutic aerosols: formulation considerations. Adv. Drug Deliv. Rev. 165-166, 127–141 (2020).
Haschek, W. M., Witschi, H. R. & Nikula, K. J. in Handbook of Toxicologic Pathology (Second Edition) (eds Haschek, W. M., Rousseaux, C. G. & Wallig, M. A.) (Academic Press, 2002).
Liu, Y., Johnson, M. R., Matida, E. A., Kherani, S. & Marsan, J. Creation of a standardized geometry of the human nasal cavity. J. Appl. Physiol. 106, 784–795 (2009).
Marshall, L. J., Perks, B., Ferkol, T. & Shute, J. K. IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component. J. Immunol. 167, 2816–2823 (2001).
Atkinson, J. J. & Senior, R. M. Matrix metalloproteinase-9 in lung remodeling. Am. J. Respir. Cell Mol. Biol. 28, 12–24 (2003).
Wolosowicz, M., Prokopiuk, S. & Kaminski, T. W. Matrix Metalloproteinase-9 (MMP-9) as a therapeutic target: insights into molecular pathways and clinical applications. Pharmaceutics 17, 1425 (2025).
Rodenburg, L. W. et al. Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis. Sci. Rep. 13, 18573 (2023).
Horowitz, A., Chanez-Paredes, S. D., Haest, X. & Turner, J. R. Paracellular permeability and tight junction regulation in gut health and disease. Nat. Rev. Gastroenterol. Hepatol. 20, 417–432 (2023).
Otani, T. & Furuse, M. Tight junction structure and function revisited. Trends Cell Biol. 30, 1014 (2020).
Gonzalez-Mariscal, L., Betanzos, A., Nava, P. & Jaramillo, B. E. Tight junction proteins. Prog. Biophys. Mol. Biol. 81, 1–44 (2003).
Fanning, A. S. & Anderson, J. M. Zonula occludens-1 and -2 are cytosolic scaffolds that regulate the assembly of cellular junctions. Ann. N. Y Acad. Sci. 1165, 113–120 (2009).
Umeda, K. et al. ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation. Cell 126, 741–754 (2006).
Vermeer, P. D. et al. MMP9 modulates tight junction integrity and cell viability in human airway epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, L751–L762 (2009).
Myc, A. et al. Nanoemulsion nasal adjuvant W(8)(0)5EC induces dendritic cell engulfment of antigen-primed epithelial cells. Vaccine 31, 1072–1079 (2013).
Bielinska, A. U. et al. Mucosal immunization with a novel nanoemulsion-based recombinant anthrax protective antigen vaccine protects against Bacillus anthracis spore challenge. Infect. Immun. 75, 4020–4029 (2007).
Bielinska, A. U. et al. Nasal immunization with a recombinant HIV gp120 and nanoemulsion adjuvant produces Th1 polarized responses and neutralizing antibodies to primary HIV type 1 isolates. AIDS Res. Hum. Retroviruses 24, 271–281 (2008).
Bielinska, A. U. et al. Distinct pathways of humoral and cellular immunity induced with the mucosal administration of a nanoemulsion adjuvant. J. Immunol. 192, 2722–2733 (2014).
Mao, X. et al. Cetylpyridinium chloride: mechanism of action, antimicrobial efficacy in biofilms, and potential risks of resistance. Antimicrob. Agents Chemother. 64, 10–1128 (2020).
Wong, P. T. et al. Formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity. Mol. Pharm. 11, 531–544 (2014).
Illum, L. Nasal drug delivery–possibilities, problems and solutions. J. Control Release 87, 187–198 (2003).
Kanno, S. et al. Benzalkonium chloride and cetylpyridinium chloride induce apoptosis in human lung epithelial cells and alter surface activity of pulmonary surfactant monolayers. Chem. Biol. Interact. 317, 108962 (2020).
Yegin, Y., Oh, J. K., Akbulut, M. & Taylor, T. Cetylpyridinium chloride produces increased zeta-potential on Salmonella Typhimurium cells, a mechanism of the pathogen’s inactivation. npj Sci. Food 3, 21 (2019).
Sharma, V. et al. Protein-surfactant interactions: a multitechnique approach on the effect of co-solvents over bovine serum albumin (BSA)-cetyl pyridinium chloride (CPC) system. Chem. Phys. Lett. 747, 137349 (2020).
Bernstein, I. L. Is the use of benzalkonium chloride as a preservative for nasal formulations a safety concern? A cautionary note based on compromised mucociliary transport. J. Allergy Clin. Immunol. 105, 39–44 (2000).
Larsen, S. T., Verder, H. & Nielsen, G. D. Airway effects of inhaled quaternary ammonium compounds in mice. Basic Clin. Pharm. Toxicol. 110, 537–543 (2012).
Kwon, D. et al. Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems. Environ. Toxicol. 34, 561–572 (2019).
George, M., Joshi, S. V., Concepcion, E. & Lee, H. Paradoxical bronchospasm from benzalkonium chloride (BAC) preservative in albuterol nebulizer solution in a patient with acute severe asthma. A case report and literature review of airway effects of BAC. Respir. Med. Case Rep. 21, 39–41 (2017).
Herriges, M. & Morrisey, E. E. Lung development: orchestrating the generation and regeneration of a complex organ. Development 141, 502–513 (2014).
Widdicombe, J. H. Early studies on the surface epithelium of mammalian airways. Am. J. Physiol. Lung Cell. Mol. Physiol. 317, L486–L495 (2019).
Parker, D. & Prince, A. Innate immunity in the respiratory epithelium. Am. J. Respir. Cell Mol. Biol. 45, 189–201 (2011).
Ioannidis, I., Ye, F., McNally, B., Willette, M. & Flano, E. Toll-like receptor expression and induction of type I and type III interferons in primary airway epithelial cells. J. Virol. 87, 3261–3270 (2013).
Golebski, K. et al. FcgammaRIII stimulation breaks the tolerance of human nasal epithelial cells to bacteria through cross-talk with TLR4. Mucosal Immunol. 12, 425–433 (2019).
Unger, W. W. et al. Nasal tolerance induces antigen-specific CD4+CD25- regulatory T cells that can transfer their regulatory capacity to naive CD4+ T cells. Int. Immunol. 15, 731–739 (2003).
Imkamp, K. et al. Gene network approach reveals co-expression patterns in nasal and bronchial epithelium. Sci. Rep. 9, 15835 (2019).
Vieira Braga, F. A. et al. A cellular census of human lungs identifies novel cell states in health and in asthma. Nat. Med. 25, 1153–1163 (2019).
Shinya, K. et al. Avian flu: influenza virus receptors in the human airway. Nature 440, 435–436 (2006).
Alves, M. P. et al. Comparison of innate immune responses towards rhinovirus infection of primary nasal and bronchial epithelial cells. Respirology 21, 304–312 (2016).
Frohlich, E. Better GRAS than sorry: excipient toxicity in pulmonary formulations. Eur. J. Pharm. Biopharm. 215, 114814 (2025).
Cao, X., Muskhelishvili, L., Latendresse, J., Richter, P. & Heflich, R. H. Evaluating the toxicity of cigarette whole smoke solutions in an air-liquid-interface human in vitro airway tissue model. Toxicol. Sci. 156, 14–24 (2017).
Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R. & Randell, S. H. Well-differentiated human airway epithelial cell cultures. Methods Mol. Med. 107, 183–206 (2005).
Gentzsch, M. et al. Pharmacological rescue of conditionally reprogrammed cystic fibrosis bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 56, 568–574 (2017).
Acknowledgements
We thank Drs. Carol Weiss and Keith Peden for comments on the manuscript and valuable discussions. This research was supported by intramural funding from the Center for Biologics Evaluation and Research (CBER), US Food and Drug Administration (FDA).
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D.A. and M.R.H.: Investigation; K.T.: Investigation and data analysis; C.D.C.: NE01, Writing—review and editing; HG: Writing—review and editing; M.Z.: Conceptualization and data analysis, Writing—original draft and editing. All authors reviewed the manuscript.
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Acosta, D., Rubel Hoq, M., Takeda, K. et al. Development of in vitro airway epithelial model to assess immune response and safety of mucosal adjuvants. npj Vaccines (2026). https://doi.org/10.1038/s41541-026-01459-z
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DOI: https://doi.org/10.1038/s41541-026-01459-z
