Flagellin from bacteria elicits a proinflammatory immune response and may act as a vaccine adjuvant. In this study, we evaluated the adjuvant effect of the N-terminus of flagellin (residues 1–99) when linked to an antigen (a truncated, conserved domain of lipoprotein E of Pasteurella multocida). Immunization of chickens with the antigen-adjuvant chimeric protein showed that the N-terminus of flagellin accelerated the antibody response and enhanced the cellular immunity (CD8⁺ T cell expansion). Stimulation of peripheral blood mononuclear cells from vaccinated chickens showed both T_H1 (IFN-γ and IL-12) and T_H2 (IL-4)-type cytokine gene expressions. In a challenge test, the N-terminus of flagellin increased the survival rate to 75%, compared to 25% in the antigen-only group. In conclusion, our study found that the N-terminus of flagellin can increase the immune response and enhance vaccine protection.

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.

Recombinant Pasterurella multocida lipoprotein E (PlpE) has been shown to protect against fowl cholera. This study aimed to determine if the signal sequence may contribute to the antigenicity and protective efficacy of recombinant PlpE. A small antigenic domain of PlpE (termed truncated PlpE, tPlpE) was constructed with (SP-tPlpE) or without (tPlpE) the signal sequence and evaluated in vitro and in vivo. In vitro, the HEK-Bule hTLR2 Cells were used to evaluate the activation of NF-kB in the test associated with the stimulation of the SP-tPlpE and tPlpE proteins. When chickens were immunized, compared to the tPlpE vaccine group, the SP-tPlpE group showed higher antibody levels and enhanced CD4⁺ T cell response. In a challenge test, the SP-tPlpE group showed a survival rate of 87.5% (n = 8), compared to 25% for the tPlpE group. It is confirmed that the inclusion of the native signal sequence enhanced protective efficacy against fowl cholera and may act as a vaccine adjuvant. The short SP-tPlpE construct is amenable to further vaccine engineering and has potential to be developed as a fowl cholera vaccine.

Introduction: Bovine respiratory disease (BRD) complex is a leading cause of economic losses in the beef and dairy cattle industries. Mannheimia haemolytica is recognised as the primary pathogen associated with this disease. While antibiotics and vaccines are widely used against it, antimicrobial resistance and limited vaccine efficacy remain obstacles. Mannheimia haemolytica leukotoxin A (LktA) has been identified as a promising candidate for subunit vaccine development against BRD. However, the low expression and biological instability of the full-length LktA complicate its production. This study evaluated the immunogenic potential of the truncated LktA protein for subunit vaccine development. Material and Methods: Truncated proteins of LktA N-terminal (nLktA) and C-terminal (cLktA) were expressed in E. coli which were small enough for stable expression yet large enough to function as effective immunogens. The immunogenicity of the recombinant truncated LktA proteins was evaluated in mouse and goat models against a phosphate-buffered saline (PBS) negative-control group. Recombinant cLktA was emulsified with oil adjuvant and used to immunise mice and goats. Results: The cLktA group had significantly higher antibody levels at four weeks post-immunisation (wpi) than the PBS group. In goats, cLktA elicited high antibody responses up to six wpi. A single administration of cLktA conferred 80% and 100% survival against a M. haemolytica challenge. Conclusion: These findings show the C-terminal region of Mannheimia haemolytica LktA to be a highly immunogenic and protective antigen and suggest its potential as a candidate for subunit vaccine development.