Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01)

Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen-specific CD8+ T-cell and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen-specific humoral immune responses against subunit protein antigens.