BSP3, a bursal-derived tetrapeptide, promotes avian B lymphocyte proliferation in vitro and enhances antigen-specific antibody responses in mice

The bursa of Fabricius is a unique central immune organ in avian species, and bioactive peptides derived from this organ play important roles in immunomodulation. However, the identification of novel, structurally simple peptides and their underlying molecular mechanisms remains to be fully elucidated. In this study, a novel tetrapeptide, designated BSP3 (bursal-derived small peptide 3; Leu-Leu-Pro-Gly, LLPG), with a molecular weight of 398.50 (m/z), was isolated from the bursa of Fabricius of White-feathered broiler chickens and identified by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS); its sequence uniqueness was confirmed by BLASTP analysis. In vivo experiments in mice showed that co-immunization with BSP3 and an inactivated H9 subtype avian influenza virus vaccine significantly enhanced serum antigen-specific IgG responses, promoted IFN-γ and IL-4 secretion, and increased antigen-stimulated splenic lymphocyte proliferation. In vitro , BSP3 promoted the proliferation of the chicken B lymphocyte line DT40 at an optimal concentration of 5 μg/mL. Exploratory transcriptome analysis of BSP3-treated DT40 cells identified 1,100 candidate genes with altered expression, which were annotated to RNA processing, actin crosslink formation, and extracellular matrix organization; MAPK signaling was positioned at the core of the pathway interaction network. RT-qPCR validation was consistent with the transcriptome data. In conclusion, BSP3 is a novel bursa-derived tetrapeptide that shows immunomodulatory activity in mice and in avian B cells. Further validation in avian hosts and functional protection studies are warranted before BSP3 can be considered a candidate vaccine adjuvant.