Adsorption sequence shapes antigen distribution and antibody binding on adjuvants in bivalent vaccines

As a critical advancement in modern immunization strategies, multivalent vaccines combine multiple antigenic components to seamlessly broaden protection against diverse pathogens within a single dose. During the formulation of aluminum-adjuvanted vaccines, antigen adsorption onto the adjuvant surface serves as a critical determinant of vaccine performance. However, it remains unclear how the sequence of adsorption during formulation influences the particle-to-particle antigen loading heterogeneity on adjuvant particles and whether such differences are associated with immune outcomes. In this study, we systematically investigate how the adsorption sequence modulates particle-to-particle antigen-loading heterogeneity and antibody binding in bivalent vaccines. Using human papillomavirus (HPV16/18) and norovirus (NoV GI.1/GII.4) virus-like particles (VLPs) as model antigens with different adsorption strengths for amorphous aluminum hydroxyphosphate sulfate (AAHS), we demonstrate that the strength of antigen–adjuvant interaction is associated with how adsorption sequence influences antigen distribution. Antigens with weaker binding affinity exhibit distinct particle-to-particle antigen loading heterogeneity depending on the formulation sequence, whereas those with strong interactions remain largely unaffected. Importantly, these variations in particle-to-particle antigen-loading heterogeneity were associated with differences in antibody and monoclonal antibody binding, and with differences in in vivo humoral immune responses. These findings highlight adsorption sequence as an important formulation parameter associated with antigen distribution and antibody responses in multivalent vaccines.