Development of a novel nanobody-fused flagellin adjuvant to enhance immunogenicity in a PCV2 subunit vaccine

Porcine circovirus type 2 (PCV2) infection causes significant economic losses in the global swine industry. The capsid (Cap) protein serves as the core antigen in PCV2 subunit vaccines, but its weak immunogenicity necessitates adjuvants to enhance the induced immune responses. Bacterial flagellin proteins are ideal candidates for next-generation vaccine adjuvants. In this study, we designed a novel flagellin-based adjuvant by co-expressing bacterial flagellin with nanobodies (Nb) specific to the PCV2 Cap protein (Nbcap-flagellin). This recombinant fusion protein leverages the high-affinity binding ability of the nanobodies to bind the Cap protein, forming an antigen-adjuvant complex to enhance vaccine immunogenicity. We further evaluated the effect of this nanobody-fusion flagellin adjuvant to enhance the immune response in mice to a subunit vaccine with PCV2 Cap protein as the model antigen. The results demonstrated that vaccination of mice with PCV2-Cap and Nbcap-flagellin adjuvant (Cap + Nbcap-flagellin) vaccine induced significantly higher levels of ELISA titers of Cap-specific antibodies, neutralizing antibodies, and immune-related cytokines compared to the PCV2-Cap vaccine without adjuvant (Cap) and the mixture of Cap protein with flagellin (Cap + flagellin). Following virulent PCV2 challenge, addition of the Nbcap-flagellin adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the serum of PCV2-challenged mice and prevented pathological changes in lymphoid tissue. These findings suggested that the Nbcap-flagellin adjuvant could enhance the immune response and provide better protection against PCV2 infection, making it a promising candidate for improving the efficacy of PCV2 subunit vaccines.