Multi-Modal Characterization of the Interactions between a Recombinant Protein Antigen and a Liposomal Adjuvant.

Subunit vaccines rely on adjuvants to enhance immunogenicity, yet antigen-adjuvant interactions in liposomal systems remain underexplored, with unknown implications that could impact the function and stability of coformulated system designs. We report a multimodal study aimed at elucidating interactions between a recombinant protein antigen, gE, and a liposomal adjuvant system, AS01B, using a commercially available vaccine as a model. Quantitative binding assays revealed no significant binding for the antigen-adjuvant pair, with only weak, dose-dependent biolayer interferometry (BLI) signals suggesting transient contacts, in contrast to the strong responses observed in positive controls. Isothermal titration calorimetry (ITC) provided clear evidence of weak, transient interactions through dose-dependent exothermic signals and characteristic saturation behavior. Ultracentrifugation, electrophoresis, and chromatographic separation techniques confirmed independent distributions for the protein and liposome components, with minor migration shifts and trace coelution under rapid flow hinting at dynamic interactions. Structural analyses by cryogenic electron microscopy (cryo-EM) and dye labeling showed negligible changes in liposome properties upon antigen mixing. These findings suggest that vaccine formulations with little or no interaction between the protein and liposome components may be stable as premixed systems and underscore the importance of employing multiple tools for characterizing multicomponent vaccines during their development.