Vaccines combining slow release and follicle targeting of antigens increase germinal center B cell diversity and clonal expansion

Vaccine adjuvants play important roles in shaping the humoral response to immunization. Here, we analyzed mechanisms of action of a clinically relevant combination adjuvant strategy, where phosphoserine (pSer)-tagged immunogens bound to aluminum hydroxide (alum) adjuvant, promoting prolonged antigen release to draining lymph nodes, are combined with a saponin nanoparticle adjuvant termed SMNP, which alters lymph flow and antigen entry into lymph nodes. When used with a stabilized HIV envelope trimer antigen in mice, this combined adjuvant approach promoted substantial enhancements in germinal center and antibody responses relative to either adjuvant alone. Using single-cell RNA and B cell receptor sequencing, we found that the alum-pSer/SMNP combination augmented the clonal expansion and diversity of the germinal center B cell repertoire, coincident with an increased proportion of S-phase germinal center B cells and expression of positive selection markers. Moreover, we found that the combination adjuvant approach, but not alum-pSer delivery or SMNP alone, promoted accumulation of intact antigen on follicular dendritic cells, reflecting integrated effects of slow antigen delivery and altered lymph node uptake. Genetic ablation of <i>Cr1/2</i> expression by follicular dendritic cells eliminated antigen accumulation and hampered the antigen-specific germinal center response, supporting antigen delivery to these cells as a key mechanism of the improved response elicited by this combination adjuvant. These results demonstrate how adjuvants with complementary mechanisms of action affecting vaccine biodistribution and kinetics can enhance humoral immunity.