Vaccinations can serve as an important preventive measure against the porcine epidemic diarrhea (PED) virus that currently threatens the swine industry. This study focuses on the development of a fusion protein vaccine, FliC₉₉-mCOE, which combines the N-terminus of flagellin (FliC₉₉) with a modified core neutralizing epitope (mCOE) of PEDV. In silico immunoinformatic analysis confirmed the construct’s non-toxic, non-allergenic, and highly antigenic nature. Molecular docking and molecular dynamics (MD) simulations demonstrated FliC₉₉-mCOE’s strong binding to the TLR-5 immunological receptor. Repeated exposure simulations and immunological simulations suggested enhanced cell-mediated immunity. Both FliC₉₉-mCOE and an inactivated PEDV vaccine were produced and tested in mice. The results from cell proliferation, ELISA, and neutralization assays indicated that FliC₉₉-mCOE effectively stimulated cellular immunity and neutralized PEDV. We conclude that the FliC₉₉-mCOE fusion protein may serve as a promising vaccine candidate against PEDV.

Flagellin activates the immune system through Toll-like receptor 5 (TLR5) and can work as an adjuvant for subunit vaccines. In this study, we tested the adjuvancy of two different N-terminal fragments of flagellin, (1) FliC₉₉, residues 1–99, and (2) FliC₁₇₆, residues 1–176, to incorporate larger areas of the hotspot region for potentially higher levels of TLR5 activation and immune response. A truncated version of the VP2 protein (name tVP2, residues 199–356) of the Infectious bursal disease virus (IBDV) was genetically linked to the flagellin constructs, and the immune response was evaluated in chickens. Results showed that both chimeric antigen–adjuvant constructs increased humoral (total IgG titers), cellular and cytokine immune response (IL-4, IFN-γ). The resulting antibody also successfully neutralized IBDV. We conclude that the N-terminus of flagellin can act as an immune activator to enhance vaccine efficacy.

Toll-like receptor 2 (TLR2) signaling is a pivotal component of immune system activation, and it is closely linked to the lipidation of bacterial proteins. This lipidation is guided by bacterial signal peptides (SPs), which ensure the precise targeting and membrane anchoring of these proteins. The lipidation process is essential for TLR2 recognition and the activation of robust immune responses, positioning lipidated bacterial proteins as potent immunomodulators and adjuvants for vaccines against bacterial-, viral-, and cancer-related antigens. The structural diversity and cleavage pathways of bacterial SPs are critical in determining lipidation efficiency and protein localization, influencing their immunogenic potential. Recent advances in bioinformatics have significantly improved the prediction of SP structures and cleavage sites, facilitating the rational design of recombinant lipoproteins optimized for immune activation. Moreover, the use of SP-containing lipobox motifs, as adjuvants to lipidate heterologous proteins, has expanded the potential of vaccines targeting a broad range of pathogens. However, challenges persist in expressing lipidated proteins, particularly within heterologous systems. These challenges can be addressed by optimizing expression systems, such as engineering E. coli strains for enhanced lipidation. Thus, lipoprotein signal peptides (SPs) demonstrate remarkable versatility as adjuvants in vaccine development, diagnostics, and immune therapeutics, highlighting their essential role in advancing immune-based strategies to combat diverse pathogens.

The nuclear factor-kappa B (NF-κB) signaling pathway plays a crucial role in regulating immune responses in epithelial cells. In this study, we established a porcine epithelial NF-κB reporter cell line (PK15-KBR) as an in vitro platform to screen plant-based extracts for their potential use as vaccine adjuvants against porcine epidemic diarrhea virus (PEDV). The NF-κB inducers were further tested for toxicity assessments, either using CCK-8 assays or intramuscular injection in mice, finally followed by vaccination studies to evaluate their adjuvancy. Initial experiments confirmed that TNF-α effectively activated NF-κB signaling in PK15-KBR cells in a dose-dependent manner, validating the platform’s reliability at Z’ value of 0.68. Of the 224 testers, 3 candidates, including chamomile, mulberry and Boerhaavia diffusa, showed induction activity; however, only chamomile induced a dose-dependent response in PK15-KBR cells. As a proof of concept, chamomile, used as an adjuvant in oral vaccination, demonstrated significantly higher IgG levels at an early stage (day 14, p < 0.05) and enhanced IgA titers. These findings highlight the use of the PK15-KBR cell line in identifying mucosal adjuvants and position chamomile extract as a promising candidate for enhancing vaccine-induced immunity.