Nano-Emulsion Incorporating Squalene and Mn²⁺ Stabilized by TA/Mn²⁺ Networks Enhances Subunit Vaccine Immunogenicity

Effective protection against infections requires humoral and cellular immune responses. Although current subunit vaccines primarily induce antibodies, they often fail to elicit strong CD8⁺ T cell responses. To overcome this challenge, we designed a dual-adjuvant nano-emulsion that integrates squalene (Sq) and Mn²⁺, using tannic acid (TA)/Mn²⁺ coordination networks for stabilization, serving as a potent immune-enhancing adjuvant system.

The ultrasonic emulsification was used to prepared nano-emulsion system (Sq@TA/Mn) combining Sq and Mn²⁺. The Sq@TA/Mn adsorbed ovalbumin (OVA) to form a Sq@TA/Mn@OVA vaccine. The cytotoxicity, ROS generation, cellular uptake, and distribution of the OVA vaccine were evaluated in DC2.4 cells. The retention of OVA vaccines in the site of injection of female C57BL/6 mice were studied using an imaging system. The mice were administered intramuscular injections of Sq@TA/Mn@OVA vaccine with prime-boost immunization strategies. The humoral immune and cellular immune responses were analysed with enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. We evaluated the nano-emulsion using a recombinant peptidoglycan-associated lipoprotein (rPal) antigen to create a Sq@TA/Mn@rPal vaccine against Acinetobacter baumannii-induced pneumonia.

The Sq@TA/Mn nano-emulsion was constructed through ultrasonic emulsification. The nano-emulsion efficiently adsorbed OVA to form a Sq@TA/Mn@OVA vaccine. The OVA vaccine exhibited a favorable safety profile, enhanced ROS generation, dendritic cell uptake, and improved antigen retention at the injection site. Compared to Alum, this vaccine enhanced the production of OVA-specific antibodies and IFN-γ, promoted the expansion of spleen effector memory T cells, and increased the population of lung-resident memory T cells.The Sq@TA/Mn adjuvant elicited higher rPal-specific IgG, IgG1, and IgG2a titers and improved the protective efficacy against infection as compared with Alum.

This study provides a novel method for the co-delivery of Mn²⁺, Sq, and antigens. These results highlighted the potential of the Sq@TA/Mn platform as a versatile and effective adjuvant for enhancing subunit vaccines.