Data are means SD. Because nearly all swine herds are seropositive for PCV2 and there are insufficient numbers of specific-pathogen-free swine, and because experimentation with swine is expensive, our results were obtained with animals other than swine. and a serum neutralization assay. The IFN- response in splenocytes harvested from immunized mice was measured by ELISA. Student’s multiple nuclear polyhedrosis virus (AcMNPV) has been used traditionally as an excellent tool to overexpress recombinant proteins in insect cells [4-6]. Because the baculovirus can enter mammalian cells and mediate the expression of transgenes under a promoter that is active in mammalian cells [7], baculoviral vectors have been exploited as versatile vaccine vehicles to produce candidate vaccines against various pathogens [8-10]. It has also been reported that a pseudotype baculovirus displaying the glycoprotein of vesicular stomatitis virus (VSV-G) on its envelope can extend the host range of the baculovirus and enhance its resistance to inactivation by animal serum complement [11-13]. Recently, AcMNPV has been further Darenzepine engineered for use as a new eukaryotic display system to express exogenous peptides on the surface of the viral envelope. This display strategy relies on thegp64 protein, which is the major envelope protein of the baculoviruses, Darenzepine which has an amino-terminal signal peptide (SP), a mature transmembrane domain (TM) and a cytoplasmic tail domain (CTD). For the surface display of exogenous peptides, a heterologous peptide was inserted between the SP and mature domain of gp64. After its expression with the native gp64, the fusion protein is translocated to the plasma membrane and incorporated into the baculoviral envelope. This method has been extended to develop pseudotyped baculoviruses as a potential vaccine delivery platform. Several research groups have Rabbit Polyclonal to C/EBP-alpha (phospho-Ser21) demonstrated that direct vaccination with pseudotyped baculoviruses can induce high titers of antigen-specific antibodies [14-16]. Based on the characteristics of the baculoviruses as a gene delivery system and surface display system together, a dual-expression-system-based recombinant baculovirus BV-GD-ORF2 was constructed in this study, which can display the PCV2 Cap protein and the VSV-G Darenzepine protein on the viral envelope and expresses the Cap protein when it is used to transduce mammalian cells. The first objective of this study was to effectively display the functional Cap protein on the baculoviral envelope, hoping that the Cap protein would retain its superior immunogenicity after in vivo immunization. Our second objective was to efficiently express the Cap protein in transduced mammalian cells. The third objective was to display the VSV-G protein on the viral envelope to boost the baculovirus resistance to complement. The potential utility of BV-GD-ORF2 as a vaccine was evaluated in a mouse model. Robust humoral and cellular immune responses were successfully induced in mice immunized with the recombinant baculovirus BV-GD-ORF2. These results suggest that a PCV2 vaccine based on the baculovirus dual expression system can be used as an alternative strategy to protect against PCV2 infection. To our knowledge, this is the first study Darenzepine to establish a PCV2 vaccine based on the baculovirus dual expression system. Results Construction of the baculovirus dual expression system The recombinant baculovirus BV-GD-ORF2 was constructed as described in the Methods (Figure?1). The infection of Sf9 cells with BV-GD-ORF2 caused in extensive cell-cell fusion (Figure?2). This phenotype is attributable to the very high expression level of VSV-G protein, which has membrane-fusion activity, under the control of the polyhedrin promoter (PPH). Open in a separate window Figure 1 Schematic representation of the structure ofBV-GD-ORF2. The ORF2 gene cassette consists of the gp64 signal sequence (SP), the ORF2 gene (ORF214C234) fused to the N-terminus of the AcMNPV major envelope protein gp64 gene (gp6425C517), and the poly(A) sequence, which was driven by a dual promoter containing the CMV immediate-early enhancer/promoter (pCMV-IE) and the polyhedrin promoter (PPH). The VSV-G expression cassette was driven by the p10 promoter (PP10). ORF214C234:ORF2 corresponding to amino acids 14C234, but lacking the N-terminal nuclear localization signal peptide of the capsid protein of PCV2. Gp6425C517: gp64 corresponding to amino acids 25C517.VSV/G: the glycoprotein of vesicular stomatitis virus. Open in a separate window Figure 2 Characterization of BV-GD-ORF2-infected Sf9 cells. Syncytium formation in Sf9 cells infected with BV-GD-ORF2 as indicated by the white arrow (a), but not in the mock-infected Sf9 cells (b). The images were captured at 72 h posttransfection. Original magnification, 100. To investigate whether the Cap protein was displayed on the membrane of BV-GD-ORF2, the purified.