Glyceraldehyde-3-phosphate Modification Promotes the Adsorption of Lysozyme onto Aluminum Hydroxide Adjuvant and Boosts Immune Responses

Hen egg-white lysozyme (HEL) was modified by glyceraldehyde-3-phosphate (G3P) to produce phosphorylated HEL (PHEL). The resulting conjugates were fractionated via cation exchange chromatography to obtain fractions with phosphate contents of 1 and 2 mol P/mol protein. Analytical ultracentrifugation (AUC) demonstrated that G3P modification induced neither degradation nor aggregation. Isothermal adsorption experiments confirmed the successful adsorption of PHEL onto aluminum hydroxide adjuvant (AH), showing a progressive increase in adsorption capacity with higher phosphate content. Fluorescence spectroscopy under ambient conditions showed no significant differences in the tertiary structure between HEL and PHEL. Similarly, circular dichroism (CD) spectroscopy indicated that G3P modification induced almost no alteration to the secondary structure of HEL, although the thermal denaturation midpoint temperature (Tm) decreased as phosphate content increased. Tandem mass spectrometry primarily identified four lysine sites involved in the reaction: K1, K13, K97, and K116. Finally, immunological assays demonstrated that G3P modification significantly enhanced the immunogenicity of PHEL. Overall, this study offers a novel strategy for enhancing antigen-adjuvant interactions and provides a robust foundation for the application of phosphorylation in vaccine development.