(T0930-04-23) Impact of a Novel Thin Film Platform on the Uptake and Permeability of Virus-Based Vaccines in an In Vitro Model of the Human Oral Mucosa

Purpose: Vaccines are a critical component of global health, yet often have limited access to low- and middle- income countries due to cost, poor thermostability and lack the infrastructure required for large immunization campaigns. To address this issue, we developed a novel thin film platform that stabilizes viruses and other therapeutic biologics for extended periods of time without refrigeration and can be utilized for immunization by the sublingual and buccal routes. The oral mucosa is densely populated with immune cells including dendritic cells (DCs), T-cells, and Langerhans cells making it an optimal target for vaccine antigen uptake [1-2]. The primary objective of this study was to use an in vitro model of the human oral mucosa to better understand the degree of viral uptake and permeability when formulated in a thin film matrix.

Methods: An air-liquid interface (ALI) transwell model using differentiated TR146 cells was utilized in these studies [3]. Transwell cultures were treated with either recombinant adenovirus 5 expressing beta-galactosidase (AdlacZ), adeno-associated virus expressing firefly luciferase (AAV), or FITC-labeled dextran of molecular weight 20 kDa (FD20). Infectious virus was determined by serial dilution on HeLa-H1 (AdlacZ) with histochemical staining and by a luciferase assay on HeLa RC32 cells (AAV). Virus genomes were quantified in media and cellular extracts by quantitative real time PCR. The concentration of FD20 was measured using a microplate reader (GloMax-Multi+ Detection System, Promega). Permeability coefficients were calculated, as defined by Fick’s Law. Transepithelial electrical resistance (TEER) was monitored with an EVOM² Epithelial Voltmeter.

Results: Transwells containing the ALI model were cultivated for 30 days and attained average TEER values of 119 ± 2.0 Ohm*cm². Histological staining indicated the presence of a significantly thicker barrier (6-fold increase) when compared to a submerged Transwell model. Twenty-four hours after treatment, 64 ± 5.6% of the total amount of AdlacZ in the film formulation (3.75×10¹⁰ ivp) was recovered in the cellular extract while 40 ± 0.7% was detected in the basolateral media. At the same time, 82 ± 0.1% of the total amount of unformulated AdlacZ (3.75×10¹⁰ ivp) was recovered in the cellular extract while 29 ± 3.1% was detected in the basolateral media. The permeability coefficients (P_app) for the formulated and unformulated AdlacZ preparations were 1.8×10^-8 and 1.6×10^-9 cm/hr, respectively. After 48 hours, 87 ± 0.9% and 80 ± 0.2% of the total amount of AAV in the film formulation and buffer, respectively, was recovered in the cellular extract. Permeability coefficients (P_app) for the formulated and unformulated AAV preparations were 2.7×10^-8 and 8.0×10^-7 cm/hr, respectively. Permeability of FD20 was measured in the presence and absence of the surfactant utilized in the film formulation. In the presence of the surfactant, the permeability coefficient was 9.5×10^-4 cm/hr with a cumulative amount of 0.18 ± 0.02 ug FD20 permeated in 24 hours. Without the surfactant, the permeability coefficient was 1.9×10^-3 cm/hr with a cumulative amount of 0.29 ± 0.04 ug FD20 permeated in 24 hours.

Conclusion: An optimized film formulation significantly improved permeability of adenovirus across the epithelial barrier. This effect was less profound with adeno-associated virus. This may be due differences in size of the virus particles (AdlacZ, ~100nm; AAV, ~25nm). Data collected with FITC-Dextran suggests that the surfactant used in the film formulation does not improve permeability of small biomolecules. Additional work with other biological drugs and small molecules with known permeabilities across the oral mucosa will be evaluated to further support these findings and validate our in vitro system as a useful tool to predict in vivo performance of film based biological drugs.

References: 1. Wu, R. Q., Zhang, D. F., Tu, E., Chen, Q. M., & Chen, W. (2014). The mucosal immune system in the oral cavity—an orchestra of T cell diversity. International journal of oral science, 6(3), 125-132. DOI: https://doi.org/10.1038/ijos.2014.48
2. Lavelle, E.C., Ward, R.W. Mucosal vaccines — fortifying the frontiers. Nat Rev Immunol 22, 236–250 (2022). https://doi.org/10.1038/s41577-021-00583-2
3. Madison M. Davis, Irnela Bajrovic, Trang Doan, Matthew Le, Maria A. Croyle. In Vitro Models of the Human Oral Mucosa for Screening Novel Formulations and Adjuvants for Film-Based Vaccines. Poster presented at: American Association of Pharmaceutical Scientists PharmSci 360 Conference, 2022 Oct 16-19; Boston, MA.

Acknowledgements: This work was supported by research grant R21 AI138017 from the National Institute of Allergy and Infectious Disease (MAC) and a David Lehr Research Award from the American Society of Pharmacology and Experimental Therapeutics (MAC). We also acknowledge support from a Dr. Feng Zhang and Dr. James McGinity Graduate Fellowship (MMD).