Enhancing secretory expression of CSFV E2 glycoprotein via signal peptide engineering in a recombinant PRRSV vector...

Porcine Reproductive and Respiratory Syndrome (PRRS) and Classical Swine Fever (CSF) are major threats to swine production, emphasizing the need for effective bivalent vaccines. Using the attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) strain vHuN4-F112 as the backbone, we generated two recombinant viruses: rPRRSV-E2 (carrying an endogenous signal peptide and transcription regulatory sequence (TRS) element) and rPRRSV-E2-△SP (signal peptide-deleted but TRS-retained). We investigated how the signal peptide modulates the expression, secretion, structural features, and immunogenicity of Classical Swine Fever Virus (CSFV) E2. Both recombinants maintained high genetic stability, exhibited parental-like plaque morphology, and stably expressed E2. The signal peptide drove efficient extracellular secretion of 52 kDa glycosylated E2, whereas rPRRSV-E2-△SP only produced an intracellular 40 kDa non-glycosylated form. Bioinformatic analysis confirmed that the signal peptide preserved all five conserved N-glycosylation sites and the overall fold of E2. In vitro assays further revealed the regulatory effects of the signal peptide on E2 secretion, glycosylation, and intracellular trafficking. In pigs, both candidates were well tolerated and induced PRRSV seroconversion by 10 days post-immunization, but only rPRRSV-E2 elicited sustained CSFV-specific antibodies. The enhanced immunogenicity likely arises from improved E2 secretion, augmented APC activation, and modulated cytokine profiles, which may counteract PRRSV-induced immunosuppression. Our findings provide a practical strategy for the co-prevention of PRRS and CSF, and offer guidance for multivalent vaccine design.