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

Vaccines incorporating slow delivery, multivalent antigen display, or immunomodulation through adjuvants have an important role to play in shaping the humoral immune response. 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 delivery to draining lymph nodes) are combined with a potent saponin nanoparticle adjuvant termed SMNP (which alters lymph flow and antigen entry into lymph nodes). When employed with a stabilized HIV Env trimer antigen in mice, this combined adjuvant approach promoted substantial enhancements in germinal center (GC) and antibody responses relative to either adjuvant alone. Using scRNA-seq and scBCR-seq, we found that the alum-pSer/SMNP combination both increased the diversity of GC B cell clones and increased GC B cell clonal expansion, coincident with increases in the expression of and the proportion of S-phase GC B cells. To gain insight into the source of these changes in the GC response, we analyzed antigen biodistribution and structural integrity in draining lymph nodes and found that the combination adjuvant approach, but not alum-pSer delivery or SMNP alone, promoted accumulation of highly intact antigen on follicular dendritic cells, reflecting an integration of the slow antigen delivery and altered lymph node uptake effects of these two adjuvants. These results demonstrate how adjuvants with complementary mechanisms of action impacting vaccine biodistribution and kinetics can synergize to enhance humoral immunity.