Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling.

Alpha hemolysin, a pore-forming toxin from Staphylococcus aureus, is a critical virulence factor for bacteria. Previous studies have demonstrated that the Hla mutant H35A (HlaH35A) serves as a potent carrier protein for subunit vaccines, yet its immunomodulatory mechanisms remain incompletely understood. Here, we demonstrate that the HlaH35A fusion enhances vaccine efficacy by targeting A Disintegrin and Metalloproteinase 10 (ADAM10) on dendritic cells (DCs), thereby activating the ADAM10-Notch signaling axis. Using the candidate antigen PA0833 from Pseudomonas aeruginosa as a model, we show that the HlaH35A-PA0833 fusion protein (HPF) significantly augments antigen uptake, DC maturation, and Notch-dependent transcriptional programs, particularly in conventional DCs (cDCs). The HlaH35A fusion drives the differentiation of Notch2-dependent cDC2s, which is marked by ESAM expression and IL-23 secretion. This process promotes Th17 and T follicular helper (Tfh) cell responses in draining lymph nodes, leading to elevated antigen-specific IgG1 titers and robust protection against acute Pseudomonas aeruginosa lung infection. Notably, ADAM10 or Notch inhibition abrogates these effects. Similarly, human monocyte-derived DCs exhibit enhanced maturation and Notch activation via the HlaH35A-ADAM10 interaction. Our findings reveal that HlaH35A is a novel carrier protein that shapes adaptive immunity by modulating cDC2 differentiation via ADAM10-Notch2 signaling, suggesting a promising strategy for Th17/Tfh-oriented vaccine design.