Protective immunity against CyHV-3 infection via different prime-boost vaccination regimens using CyHV-3 ORF131-based DNA/protein subunit vaccines in carp Cyprinus carpio var. Jian.

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1β, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.