The purified active fraction of Albizia julibrissin saponin (AJSAF) is an ideal 
adjuvant candidate that improves antigen-specific both cellular and humoral 
immune responses and elicits mixed Th1/Th2 responses, but its mechanisms remain 
unclear. The key features of action of AJSAF were investigated in mice immunized 
with Newcastle disease virus-based recombinant influenza vaccine (rL-H5) and 
AJSAF at the same leg (AJSAF+rL-H5) or different legs (AJSAF/rL-H5). The 
adjuvant activity of AJSAF on rL-H5 is strictly dependent on their spatial 
colocalization. Serum H5 antigen (H5Ag)-specific IgG, IgG1, IgG2a, and IgG2b 
antibody titers in AJSAF+rL-H5 group were significantly higher than those in 
AJSAF/rL-H5 group. The mechanisms of selectivity of Th1 or Th2 in mice induced 
by AJSAF was explored by the transcriptomic and proteomic profiles of 
H5Ag-stimulated splenocytes from the immunized mice using gene microarray and 
two-dimensional difference gel electrophoresis coupled with matrix-assisted 
laser desorption/ionization time-of-flight mass spectrometry. Compared to rL-H5 
alone, AJSAF/rL-H5 induced more differentially expressed genes (DEGs) than 
AJSAF+rL-H5, whereas AJSAF+rL-H5 upregulated higher mRNA expression of Th1 
(T-bet, IFN-γ, TNF-α, IL-12β, and IL-12Rβ1) and Th2 (IL-10 and AICDA) immune 
response genes. The neutrophil response and its derived S100A8 and S100A9 might 
be involved in the AJSAF-mediated Th1 response. Meanwhile, AJSAF might induce 
the adaptive immune responses by improving a local innate immune 
microenvironment. These findings expanded the current knowledge on the 
mechanisms of action of saponin-based adjuvants, and provided new insights into 
how adjuvants shape adaptive immune responses.