Purpose: Virosomes have been developed as an efficient vaccine delivery system. Virosomes are spherical, unilamellar lipid-based carriers, intercalated with functional glycoproteins to reflect the natural virus, however the lack of viral RNA means there is no risk of infection. In this work, we have developed a method to produce a dry powder form of an HIV-1 vaccine based on influenza-derived virosomes using spray-drying. Formulations have been optimised for oral and nasal delivery, with the aim of using the enhanced stability of the dry powder formulation to provide cold chain independent shipping and storage.
Methods: Influenza virosomes were manufactured with intercalated influenza hemagglutinin (HA) (A/H1N1), a recombinant protein fragment (rgp41) and a synthetic lipopeptide (P1) both derived from the HIV gp41 protein. Formulation excipients were trehalose, sodium alginate and L-leucine. Spray dried powders were produced using a Buchi B-290 spray dryer with a high-performance cyclone. Air was used as the drying medium. The inlet temperature was adjusted to achieve an outlet temperature of 60 and 80°C. Virosomes, pre and post spray drying were characterised for particle size (DLS, NanoSight, Malvern) and for the presence of intact protein structure (SDS-page electrophoresis). Powder particle size was measured using laser diffraction (HELOS, Sympatec). In vivo pre-clinical testing was performed in a rodent and rabbit models for dose delivery (gamma scintigraphy) and immunogenicity.
Results: The virosome stock solution was spray dried at 60°C in a 10% w/v feed solution containing either a 99:1 trehalose:leucine mixture for oral delivery or trehalose:alginate 90:10 for nasal delivery. The hydrodynamic diameter of the virosomes, measured by DLS before and after spray drying shows that the virosomes remained intact. Stock and spray dried virosomes were also analysed by SDS page electrophoresis, using a 4-12% Bis Tris gel plate and non-reducing conditions. Results showed that the functional proteins marked in the stock virosomes, remained present after spray drying, and by western and spot blots they are recognized by specific antibodies. The particle size of powders manufactured was typically between 2.5 – 4.0 µm for oral delivery, and > 10 µm for nasal delivery. In vivo imaging by gamma scintigraphy showed that the nasal powders were well distributed in the nasal cavity, and that nasal administration could generate immune responses comparable to subcutaneous administration following three dose occasions over 56 days.
Conclusion: Virosomes were successfully spray dried into three dry powder formulations, designed for oral and nasal drug delivery. The spray drying process was demonstrated to successfully maintain the integrity of the virosomes and vaccinal antigens, and the physical properties of the powder could be tuned for the different delivery routes. An enteric-coated oral capsule and a single dose nasal spray have been developed. In vivo testing showed the nasal delivery route was effective at generating a comparable immune response to subcutaneous administration.
Richard A. Johnson– CEO, Upperton Pharma Solutions, Nottingham, England
Laura Mason– Upperton Pharma Solutions
Jack Sorrell– Upperton Pharma Solutions
Andrew Naylor– Upperton Pharma Solutions, Upperton Ltd.
Mario Amacker– Mymetics Corporation
Toon Stegmann– Mymetics Corporation
Sylvain Fleury– Mymetics Corporation
Richard Johnson– Director, Upperton Limited, Nottingham