(T1530-05-26) In Vitro Models of the Human Oral Mucosa for Screening Novel Formulations and Adjuvants for Film-Based Vaccines

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. While 6 adjuvants have been approved for human use, none currently exist for mucosal administration. The primary objective of this study was to assess the utility of several models of the human oral mucosa for development of novel formulations and adjuvants for film-based vaccines

Methods: Two human oral explant models (partial and full-thickness, MatTek) and differentiated TR146 cells (airlifted and submerged Transwell configurations) were utilized in these studies [1]. Each model was treated with live human influenza virus (A/Puerto Rico/8-9VMC3/1934 (H1N1)) or recombinant adenovirus 5 expressing the beta-galactosidase transgene (AdlacZ) (Table 1). Infectious virus was determined by serial dilution on MDCK (influenza) and HeLa-H1 cells (adenovirus) with histochemical staining. Virus genomes were quantified in media and tissue samples by quantitative real time PCR. Permeability coefficients for each virus were calculated, as defined by Fick’s Law. Explants were also treated with TRP agonists cinnamaldehyde (0.01 µM), vanillin, icilin (100 µM), and eugenol (100 nM). Cytokine release in the basolateral chamber of explants were quantified with the Th1/Th2 Cytokine 11-Plex Human ProcartaPlex™ Panel (Invitrogen). Transepithelial electrical resistance (TEER) for each sample was monitored with an EVOM² Epithelial Voltmeter.

Results: The partial thickness model consists of 8-11 human-derived oral epithelial cell layers from human donors. The full-thickness model has an additional basolateral collagen matrix and integrated antigen-presenting cells (APCs). After 3 hours, 84 ± 0.2% of the total amount of influenza in the low dose film formulation was detected in the basolateral chamber of the partial thickness model while 75 ± 1.8% of the total amount of the high dose preparation was detected at the same time point. Ninety-six percent (± 7.2%) of the total amount of unformulated virus remained in the tissue. TEER values for this model fell from 410 ± 50 to 330 ± 80 ohm*cm² at the end of this study. After 24 hours, films containing TRP activators increased IL-1β cytokine response 50-fold relative to films lacking TRP agents and unformulated virus. IL-6 cytokine production was not significantly different across explants treated with TRP activators, formulated and unformulated virus, and had an average basolateral concentration of 140pg/mL after 24 hours. After 24 hours, 72 ± 0.4% of the total amount of adenovirus in the high dose film formulation was detected in the tissue of the partial thickness model while 71 ± 0.5 and 67 ± 0.6% was found in the tissue after treatment with the medium and low dose formulations, respectively. TEER values fell from 2375 ± 55 to 975 ± 47 ohm*cm² at the 24-hour time point. Adenovirus permeability in the full thickness model was not significantly different than that seen in the partial thickness model as 75 ± 1, 71 ± 0.1 and 69 ± 2.5% of the high, medium, and low doses were found in tissues 24 hours after treatment.  Mass balance calculations revealed that the remaining virus for each dose was found in the basolateral medium within 2 hours. TEER values remained constant during the 24-hour time point (351 ± 28 ohm*cm², t=0 vs 375 ± 54 ohm*cm² t=24h). An optimized film formulation increased transduction efficiency 2-fold in undifferentiated TR146 cells. Preliminary studies in the Transwell model indicate the submerged model develops TEER values of 134 ± 13 ohm*cm² by day 27 while the airlift model reached TEER values of 120 ± 7 ohm*cm² by day 35.

Conclusion: Barrier integrity, physical properties of viruses, and an optimized film formulation play a key role in permeability through the oral mucosa or vector uptake by the oral epithelium for antigen expression. Transient receptor potential (TRP) channels in the oral mucosa that play a role in cytokine release and activation of antigen presenting cells are possible targets for novel adjuvants [2].

References: 1. Grace C. Lin, Tamara Leitgeb, Alexandra Vladetic, Heinz-Peter Friedl, Nadine Rhodes, Angela Rossi, Eva Roblegg & Winfried Neuhaus (2020) Optimization of an oral mucosa in vitro model based on cell line TR146, Tissue Barriers, 8:2, 1748459, DOI:10.1080/21688370.2020.1748459
2. Santoni G, Morelli MB, Amantini C, Santoni M, Nabissi M, Marinelli O, Santoni A. "Immuno-Transient Receptor Potential Ion Channels": The Role in Monocyte- and Macrophage-Mediated Inflammatory Responses. Front Immunol. 2018 9:1273. doi: 10.3389/fimmu.2018.01273

Acknowledgments: 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 PhRMA Foundation (IB) and AFPE Predoctoral Fellowships (MDL).

Table 1: Doses used for each study.