A novel Alhagi honey polysaccharide-Alum Pickering emulsion adjuvant enhances systemic and mucosal immunogenicity of a BVDV vaccine in calves

Mucosal surfaces are the primary barrier to infection. For pathogens like bovine viral diarrhea virus (BVDV) that invade via the intestinal mucosa, vaccines capable of inducing mucosal immunity are crucial for mounting a comprehensive immune response. In this study, a new assembled Pickering emulsion (AHPPE) was created. This emulsion was constructed by employing an Alum adjuvant (Alum) loaded with Alhagi honey polysaccharide (AHP)-a known enhancer of intestinal mucosal immunity-as the shell, and squalene containing all-trans retinoic acid (RA), an agent targeting intestinal mucosa, as the core, utilizing ultrasonic emulsification techniques. The mucosal and systemic immune responses induced by this formulation as an adjuvant for a BVDV vaccine were evaluated. Results showed that AHPPE microparticles were raspberry-shaped (~ 2000 nm) with a positive surface charge, efficiently loaded BVDV antigens, and remained stable for > 30 days. In calves, AHPPE-adjuvanted immunization significantly elevated serum BVDV-specific IgG and fecal specific IgA ( P < 0.05). Serum levels of IL-10, CCL28, and CCR9 proteins were also significantly increased at peak response. In vitro, AHPPE was efficiently internalized by monocyte-derived dendritic cells (MoDCs). Transcriptomic analysis identified upregulated expression of genes linked to TLR signaling, MHC II presentation, and chemokine activity. Collectively, these in vitro findings suggest that AHPPE enhances systemic humoral immunity and promotes an immune profile associated with key mucosal correlates, with transcriptomic data pointing to potential involvement of chemokine network modulation. These observations are hypothesis-generating and await functional validation. In conclusion, AHPPE is a novel Pickering emulsion adjuvant that effectively enhances sys1temic and mucosal BVDV-specific antibody responses in calves. These findings establish AHPPE as a promising immunostimulatory candidate to improve the immunogenicity of inactivated BVDV vaccine, warranting further evaluation in viral challenge models. The concurrent modulation of gut-homing chemokines provides a mechanistic correlate for its mucosal immunomodulatory activity. Protective efficacy, however, awaits confirmation in future challenge studies.