A n-heneicosane solid emulsion as a single-dose subunit vaccine elicits broad and durable immune responses in an aged and a Pox virus model.

The development of single-dose vaccines capable of inducing durable humoral and cellular immune responses would eliminate the need for additional booster doses, which is particularly important for resource-limited settings or in a pandemic scenario. Here, we report the development of a sustained-release n-heneicosane solid emulsion designed to enhance subunit vaccine immunogenicity after a single administration. Fast- and slow-release emulsions were formulated by varying surfactant content and adding CpG to increase the adjuvant activity. The emulsions were characterized by release kinetics, cytotoxicity, and innate immune activation in dendritic cells. The slow-release formulation demonstrated prolonged antigen release and promoted pro-inflammatory cytokine production without inducing significant cytotoxicity. In vivo, a single subcutaneous immunization with antigen-loaded n-heneicosane emulsions elicited robust and long-lasting antigen-specific antibody responses and cellular immune responses in mice. A Sustained IgG, IgG1 and IgG2c responses was observed for 165 days after a single immunization, while an IFN-γ and IL-2 cellular response in splenocytes was observed after 180 days post-immunization. In an Orthopoxvirus subunit vaccine model, the emulsion platform induced high levels of IgG, IgG1 and IgG2a antibody responses at higher or comparable levels than a prime-boost immunization with the MF-59-like adjuvant Addavax. Finally, strong humoral and cellular responses were also generated following a single immunization in 18-month-old mice, highlighting the potential of this platform to overcome age-associated immune dysfunction. Overall, these findings demonstrate that n-heneicosane emulsions function as an effective single-dose vaccine delivery system capable of promoting durable adaptive immunity across multiple subunit vaccine models and aged groups.