Outer membrane vesicle loaded thin films as a stable room temperature vaccine platform

The thermostability of vaccines is paramount to their efficacy, but many vaccines are highly sensitive to temperature fluctuations and depend on cold-chain systems to maintain their immunogenicity. Outer membrane vesicles (OMVs), being derived from bacteria and containing multiple protein antigens on their surface, are no exception, and must be stored frozen long term. As such, there is a need to increase the thermostability of OMVs in order to improve access to OMV-based vaccines across the globe. Furthermore, formulation of OMVs into a platform capable of being administered via the mucosa would improve immunogenicity, as the mucosa is the main site of pathogen infection for many diseases. Here, we formulated OMVs into thin films containing various excipients and evaluated the stability of the OMV structure and antigen proteins within the OMVs over 8 weeks. Thin film compositions were optimized based on film formation ability, then four formulations were chosen and tested for OMV recovery. PMAL, an amphiphilic compound often used to stabilize biomolecules, impacted the OMVs the most, degrading their structure within 4 weeks. Other film components, including polymers and sugar alcohols, did not degrade the OMVs after 8 weeks, indicating that these compounds maintain the OMV structure in the long term. Antigen proteins on OMVs remained most stable in films containing PEG and glycerol. Films were also evaluated for their dissolution in mucosa-mimicking media, with films dissolving in less than 5 min in most solvents, demonstrating applicability as a buccally administered vaccine platform.