Multi-copy expression of classical swine fever virus E2 in a recombinant pseudorabies virus enhances immunogenicity...

Classical swine fever virus (CSFV) and pseudorabies virus (PRV) remain significant threats to the swine industry. Although recombinant PRV vectors represent promising platforms for bivalent vaccine development, their application is frequently limited by insufficient expression of heterologous antigens, which may compromise protective efficacy. To address this limitation, we employed a multi-copy expression strategy to enhance CSFV E2 protein levels in a PRV-based vector. Using CRISPR/Cas9-mediated gene editing, we constructed recombinant PRVs expressing one, two, or three copies of the E2 gene by sequential insertion into the gE/gI, tk, and gG loci. In vitro validation demonstrated increased detectable E2 expression in the multi-copy recombinant viruses. Meanwhile, the recombinant viruses maintained virion morphology and replication kinetics comparable to those of the parental strain PRV-GX. Immunogenicity studies in rabbits showed that PRV-3CE2 elicited stronger E2-specific humoral responses and E2-associated cytokine recall responses than PRV-2CE2, while both recombinant viruses induced detectable neutralizing activity. In challenge experiments, although vaccination did not completely prevent febrile responses following CSFV challenge, both PRV-2CE2 and PRV-3CE2 reduced CSFV RNA loads in blood compared with the mock group, suggesting partial protective efficacy. Additionally, both PRV-2CE2 and PRV-3CE2 provided complete protection against lethal PRV challenge in rabbits. These findings suggest that multi-copy E2 expression enhances E2-associated immunogenicity and supports further optimization and evaluation of PRV-3CE2 as a PRV-CSFV bivalent vaccine candidate.