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

Vaccines incorporating slow release, multivalent antigen display, and/or immunomodulation through adjuvants have important roles 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 release 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 augmented the clonal expansion and diversity of GC B cell repertoire, coincident with increased expression of positive selection markers and 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 intact antigen on follicular dendritic cells (FDCs), reflecting an integration of the effects by slowing antigen release and altering lymph node uptake. Ablating the Cr1/2 complex on FDCs significantly hampered antigen-specific GC response, corroborating enhanced FDC decoration as the key mechanism of improved vaccine response. These results demonstrate how adjuvants with complementary mechanisms of action impacting vaccine biodistribution and kinetics can synergize to enhance humoral immunity.