Parenteral neonatal priming followed by heterologous mucosal booster favors IgM+ memory B cell induction over systemic plasma cell differentiation

Early-life vaccinations often yield short-lived antibody (Ab) responses due to limited germinal center (GC) reaction and plasma cell (PC) survival. The aim of the study was to assess how vaccine delivery routes (homologous versus heterologous) in mice shape early-life GC dynamics and outputs. 14 days post-booster immunization, spleen, cervical (CLN), and inguinal (ILN) lymph nodes were analyzed for GC B cells, T follicular helper (TFH) and regulatory (TFR) cells, and GC-derived memory B cells and expression of B cell activating factor receptor (BAFF-R)/transmembrane activator and cyclophilin ligand interactor (TACI) on GC and memory B cells. Plasmablast/PC and their B cell maturation antigen (BCMA) expression, vaccine-specific Ab-secreting cells (ASCs), and serum and salivary Abs were also assessed. We observed that the booster route determined the anatomical site of the GC responses. Homologous subcutaneous (s.c.)/s.c. immunization increased GC induction in spleen and s.c. draining LNs, the ILNs, whereas s.c./intranasal (i.n.) immunization primarily induced GCs in mucosal draining LNs, the CLNs. The memory B cell composition was also route-dependent, with s.c./i.n. immunization preferentially generating GC-derived IgM+ memory B cells across lymphoid tissues. In contrast, homologous s.c./s.c. immunization promoted PC differentiation in spleen, yielding more BCMA+ cells especially in ILNs and BM, associated with elevated vaccine-specific serum IgG (days 7-14) and increased IgG ASCs in spleen and bone marrow. Heterologous s.c./i.n. immunization instead favored PC differentiation only in the CLNs and elevated serum and salivary IgA Abs. Correspondingly, BAFF-R and TACI expression was elevated on splenic GC B cells following s.c./s.c. immunization, whereas a higher expression was observed on CLNs GC B cells and IgM+ GC-derived memory cells after s.c./i.n. immunization. Together, these findings demonstrate that the booster immunization route directs the anatomical site of GC activity and determines the dominant GC-derived memory B cell subset. Homologous s.c./s.c. immunization maximizes systemic IgG responses, through enhanced BCMA-associated PC survival, whereas s.c./i.n. immunization promotes IgM+ GC-derived memory B cell induction while confining PC differentiation and BCMA expression to CLNs, resulting in combined systemic and mucosal Ab responses. These results support rational, route-informed early-life vaccine design to selectively enhance systemic IgG or combined systemic–mucosal antibody responses.