Protective efficacy of a recombinant pseudorabies virus expressing three copies of PCV2 Cap protein and its ability to decrease PCV2 loads in pigs

Porcine circovirus type 2 (PCV2) is one of the major pathogens causing significant economic losses in the global swine industry. The commercial vaccines are widely used to control this virus infection but fail to completely eliminate PCV2. Pseudorabies virus (PRV) has been used as a vector to develop a bivalent vaccine against PCV2 and PRV. However, no commercial live vaccines based on the PRV vector are currently available due to their low protective efficacy against PCV2. In this study, we constructed four recombinant viruses, ZJ01-CAP/CP1 to CP4, by sequentially deleting TK, gG, and US9/US2 genes and inserting PCV2 Cap gene with different promoters, respectively, by using a variant PRV strain ZJ01 with deletions of gI/gE genes. Among them, ZJ01-CAP/CP4, which contains Cap insertions at three distinct loci, exhibited high genetic stability and comparable growth kinetics to the parental strain. In both mice and piglets, ZJ01-CAP/CP4 elicited robust humoral and cellular immune responses, characterized by high levels of neutralizing antibodies and significant secretion of IL-4 and IFN-γ. Vaccination with ZJ01-CAP/CP4 provided complete protection against lethal challenge with the variant PRV ZJ01 strain and significantly reduced viral loads and pathological lesions following PCV2 challenge. Notably, in a therapeutic model, vaccination of the pigs pre-infected with PCV2 accelerated the clearance of the virus from the blood and tissues. These findings suggest that ZJ01-CAP/CP4 is a promising bivalent vaccine candidate that offers dual protection and aids in the accelerated clearance of PCV2 in infected herds.IMPORTANCEPorcine circovirus type 2 (PCV2) is one of the major pathogens causing huge economic losses to the global swine industry. The current commercial vaccines fail to eliminate PCV2 infection. Pseudorabies virus (PRV) has been used as a vector to develop bivalent vaccines against PCV2 and PRV. But there is no commercial recombinant PRV-PCV2 live vaccine because of low protective efficacy against PCV2. Here, we developed a novel recombinant PRV ZJ01-CAP/CP4 with multi-locus PCV2 Cap insertion with different promoters, and six virulence genes deletion, which confers safer and more robust dual protection against PRV and PCV2. Furthermore, ZJ01-CAP/CP4 uniquely accelerates PCV2 clearance in pre-infected pigs. It provides a promising bivalent vaccine candidate and a new strategy for PRV-based multivalent vaccine development, with important clinical implications for swine disease control.