Intrinsic Adjuvant Effect of Lipoprotein Signal Peptide in a Recombinant PEDV Subunit Vaccine

The Porcine Epidemic Diarrhea Virus (PEDV) spike (S) protein is a key glycoprotein that mediates viral binding and entry, making it a prime target for subunit vaccine development. Within its S1 domain, the core neutralizing epitope (COE; residues 499–799) is rich in predicted B- and T-cell epitopes as well as neutralizing determinants, underscoring its strong immunogenic potential. This study explores whether fusing a bacterial lipoprotein signal peptide to the COE domain can enhance its antigenicity and protective efficacy by promoting lipidation-driven Toll-like receptor 2 (TLR2) activation. Two constructs were generated: COE with signal peptide (SP-COE) and COE without signal peptide. Their performance was assessed using in silico, in vitro, and in vivo approaches. Computational analyses confirmed broad B- and T-cell epitope coverage, stable structural models, and favorable receptor binding. In vitro, HEK-Blue hTLR2 reporter assays showed that SP-COE triggered significantly higher NF-κB activation compared to COE, validating enhanced TLR2 engagement. In vivo, SP-COE vaccination in mice resulted in stronger humoral and cellular immunity, evidenced by higher total IgG and virus-neutralizing antibody titers, robust cytokine responses (IFN-γ, IL-4), and increased CD4⁺ T-cell activation. Collectively, these findings indicate that incorporating a lipoprotein signal peptide augments the immunogenicity and protective performance of the PEDV COE antigen, supporting its role as a built-in adjuvant for rational subunit vaccine design.