Rational Development of a Novel Emulgel Adjuvant for Single‐Shot Effective Vaccination: A Multivariate Analysis Approach

Nanoemulsions, like MF59, are potent vaccine adjuvants due to their immunogenicity, scalability, stability, and generation of broad cross-clade neutralizing immune responses, facilitating their feasibility for pandemic preparedness. However, they are unsuitable for single-shot applications due to limited immunogenicity with subunit antigens, and need to be explored for key immunogenic formulation variables like surfactant type, antigen anchoring, oil-globule solidification, hydrogel polymer trapping, or globule size. Here, a multivariate analysis approach is used for the first time in adjuvant development, selecting 14 formulations from over 150, each with a unique formulation variable, to evaluate their immunological properties on C57BL/6 mice. Results show that nanoemulsions with hydrogel trapping, smaller globule size, or mannide monooleate cosurfactant significantly enhance immunogenicity, each by 5-to-10 folds over MF59. For the first time, these formulation variables are combined into an "optimized emulgel" that is predicted to maximize immunogenicity after a single shot, model fit gave (R² = 0.97). The optimized emulgel triggers long-lasting immune responses matching both the model's prediction and those triggered by the gold-standard Freund's adjuvant against various antigens, while maintaining excellent safety. The findings highlight the potential of emulgels as a novel adjuvant class and underscore the utility of multivariate analysis in adjuvant design.