Lyophilizable Thermostable Nano-Aluminum Adjuvant and Combinations Induced Robust Antigen-Specific Humoral and Cellular Immunity in Mice

Aluminum adjuvants have been used in licensed human vaccines for almost a century. However, they can neither induce an efficient cellular immune response, nor be frozen or lyophilized due to aggregation and diminished Adjuvant efficacy. This study developed a series of lyophilizable nano-aluminum adjuvants by coating nanoscale aluminum Adjuvant (Adju-Phos) with glycol chitosan (GCS, 20 kDa) using microfluidics technology. When the GCS/alum weight ratio was higher than 4:1, the nano-aluminum adjuvants withstood autoclaving and freeze-drying treatments in the presence of trehalose without significant physicochemical changes. Moreover, the lyophilized nano-aluminum adjuvants remained stable for 15 months at room temperature. When combined with HPV16 L1 virus-like particles (VLPs), the nano-aluminum Adjuvant with 7:1 GCS/alum weight ratio (G7-A1) elicited stronger adaptive immunity, as evidenced by higher neutralizing antibodies, balanced IgG1/IgG2C ratio, elevated IFN-γ/TNF-α/IL-4/IL-10 secretion, and increased Th1/CTL/memory T cell frequencies. Furthermore, combining G7-A1 with MF59-like emulsion or C-di-AMP enhanced antigen-specific humoral and cellular immunity, memory T cell responses, and dendritic cell activation. These findings demonstrate the great potential of nano-aluminum adjuvants in improving vaccine efficacy, and suggest that they may serve as alternatives suitable for vaccines requiring balanced Th1/Th2 immunity or strong cellular immunity.