Category: Formulation and Quality
Purpose: Previously, a live recombinant adenovirus-based vaccine against Ebola was successfully stabilized in an optimized thin film (1). This formulation protected primates from Ebola after intranasal immunization (2). We aim to identify physiochemical determinants of viral stability and how they impact the immunogenicity of vaccines administered by the oral route in this novel thin film matrix. The hypothesis tested is that interactions between a zwitterionic surfactant and virus capsid proteins are key for preserving virus stability during storage and in the physiological environment of the mouth.
Methods: Adenovirus expressing beta-galactosidase and H1N1 influenza virus (BEI Resources) were utilized to create films at ambient temperature. FTIR, XRD, and TEM assessed crystallinity and virus: excipient interactions. Recovery of infectious virus from film was evaluated by an infectious titer assay. Anesthetized BALB/c mice were immunized by the SL and BU routes with films containing (2000 CEID50) H1N1 virus. Anti-influenza antibody titers were determined by neutralizing and ELISA assays of serum collected 28d later.
Results: No significant difference between FTIR spectra in the 4000–800 cm-1 region was observed for films prepared with and without adenovirus except for a 2-fold increase at wavenumber 3300. XRD confirmed that optimized films exhibited a single, broad, low intensity peak. TEM revealed virus suspended in excipients with entire particles encapsulated within micelle-like structures. Addition of glutamic acid (GA) that binds to adenovirus capsids reduced infectious titer by 64% after 7 days at 20ºC. Virus titer did not drop in films stored under the same conditions in the absence of GA. Mice immunized by the SL and BU routes had anti-influenza neutralizing antibody titers (1:640, p >0.5)) and IgG levels similar to that of mice immunized by IM injection (0.240, p > 0.5).
Conclusion: FTIR and glutamic acid saturation studies revealed that N-H bonds between the positively charged arm of the surfactant and glutamic acid residues on hexon proteins were critical for maintaining stability during long term storage (3). They may also be responsible for maintaining stability in the oral mucosa allowing a influenza vaccine to produce antibody levels after SL or BU administration which was comparable to IM injection, making this platform viable for oral immunization campaigns.
References: (1) Bajrovic, I., et. al. Sci Adv. 2019. In Review. (2) Choi, J.H. et. al. Mol. Pharm. 2015. 2712–2731. (3) Karlin S, Brendel V. PNAS 1988. 9396-9400.