To understand requirements for immunization via the oral mucosa, an in vitro model that recapitulates the physical barrier of the mouth, allows for quantification of antigen uptake and permeability and mounts an inflammatory response to antigen and adjuvant is needed. The physical structure of 4 models of the human oral mucosa was determined by histochemical staining and transepithelial electrical resistance (TEER) measurements. A TR146 based air-liquid interface (ALI) model most closely mimicked in vivo conditions. This was confirmed by validation studies using dextran and caffeine as diffusant molecules. Apparent permeability coefficients (Papp) of adenovirus (Ad) and adeno-associated virus (AAV) in this model were 4.3 × 10-13 and 2.2 × 10-10 respectively, while 100% of the total dose of H1N1 influenza remained in the epithelial layer. Sodium glycocholate and a hyperosmotic formulation improved the amount of Ad (p = 0.02) and AAV (p = 0.003) that entered the epithelium, respectively. Significant amounts of IL-6 (45.1 pg/mL), GM-CSF (94.7 pg/mL) and IFN-γ (4.3 pg/mL) were produced in response to influenza infection. Treatment with an AS03-like adjuvant induced production of IL-6 (34.9 pg/mL), TNF-∝ (43 pg/mL), GM-CSF (121.2 pg/mL) and IFN-γ (14.1 pg/mL). This highlights the contribution of differentiated epithelial cells to the immune response to vaccines and adjuvants.

Adeno-associated virus (AAV) vectors are stored and shipped frozen which poses logistic and economic barriers for global access to these therapeutics. To address this issue, we developed a method to stabilize AAV serotype 9 (AAV9) in a film matrix that can be stored at ambient temperature and administered by systemic injection. AAV9 expressing the luciferase transgene was mixed with formulations, poured into molds and films dried under aseptic conditions. Films were packaged in individual particle-free bags with foil overlays and stored at various temperatures under controlled humidity. Recovery of AAV9 from films was determined by serial dilution of rehydrated film in media and infection of HeLa RC32 cells. Luciferase expression was compared to that of films rehydrated immediately after drying. Biodistribution of vector was determined by in vivo imaging and quantitative real-time PCR. Residual moisture in films was determined by Karl Fischer titration. AAV9 embedded within a film matrix and stored at 4 °C for 5 months retained 100% of initial titer. High and low viscosity formulations maintained 90 and 85% of initial titer after 6 months at 25 °C respectively. AAV was not detected after 4 months in a Standard Control Formulation under the same conditions. Biodistribution and transgene expression of AAV stored in film at 25 or 4 °C were as robust as vector stored at −80 °C in a Standard Control Formulation. These results suggest that storage of AAV in a film matrix facilitates easy transport of vector to remote sites without compromising in vivo performance. Adeno-associated viruses (AAVs) are small viruses that are used to deliver medicines and vaccines. Prior to administration, they are stored in freezers set to very low temperatures and must be discarded if they thaw during transportation to clinics. AAV was embedded in a film to protect the virus during transportation and storage. The virus remained stable for 6 months at room temperature and during shipment from Texas to North Carolina. The ability to store and transport AAV without the need for complex packaging and temperature control will increase global access to vaccines and other medicines that use AAVs for delivery. Doan et al. characterize and assess the stability of film matrix embedded Adeno-Associated Virus 9 (AAV9) vectors during storage and transport at ambient temperatures. High and low viscosity formulations of AAV9 stored at 25 °C maintain titer for 6 months.

Temperature-stable dissolving film eliminates cold-chain storage and successfully immunizes mice sublingually and buccally. A novel, thin-film platform that preserves live viruses, bacteria, antibodies, and enzymes without refrigeration for extended periods of time is described. Studies with recombinant adenovirus in an optimized formulation that supports recovery of live virus through 16 freeze-thaw cycles revealed that production of an amorphous solid with a glass transition above room temperature and nitrogen-hydrogen bonding between virus and film components are critical determinants of stability. Administration of live influenza virus in the optimized film by the sublingual and buccal routes induced antibody-mediated immune responses as good as or better than those achieved by intramuscular injection. This work introduces the possibility of improving global access to a variety of medicines by offering a technology capable of reducing costs of production, distribution, and supply chain maintenance.

As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4 × 109 infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0 × 1010 ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.