| Baculovirus is an enveloped DNA virus capable of infecting insects in nature. Baculovirus can insert as large as 38 kb foreign gene and can drive transgene expression under the control of appropriate promoter, thus eliciting immune responses in vivo; Meanwhile, baculovirus cannot replicate in mammalian cells, hence easing the safety concerns.In addition, baculovirus itself can act as an adjuvant by stimulating innate immunity. Therefore, baculovirus has the potential to be served as an ideal candidate vaccine vector, and it has been applied in gene engineering vaccine development to prevent many animal diseases.Despite these advantages, there are still two major obstacles in the use of baculovirus system for animal vaccine. One major obstacle is its relatively low expression yield, which leads to the improvement of production costs, therefore making the baculovirus-based animal vaccine relatively expensive and slowing their widespread applications in many farms. The other major obstacle is the inactivation of baculovirus by serum complement. Therefore, to overcome these problems associated with traditional baculovirus system and develop a safe and effective baculovirus-based animal vaccine, we studied the baculovirus system through three aspects and constructed a novel baculovirus vector vaccine using CSF as an animal disease model to evaluate immune response. The main results were shown as below:1. Based on the mechanism to increase protein expression of the translational enhancer, three translational enhancers including Syn21(a synthetic AT-rich 21 bp sequence), P10UTR(the 3’-untranslated region from the baculovirus p10 gene) and IVS(intron from Drosophila myosin heavy chain gene) were selected to optimize the conventional baculovirus vector using GFP as a reporter gene. The results showed that Syn21 increased expression of GFP by a factor of 4.04 ± 0.12(n = 3); P10 UTR increased expression of GFP by a factor of 1.35 ± 0.02(n = 3); while IVS decreased expression of GFP by a factor of 0.65 ± 0.02(n = 3). The results also showed that Syn21 and P10 UTR have synergistic effects and increased the GFP yield by a factor of 4.80 ± 0.08(n = 3). Based on the results above, we asked whether the synergistic effects on gene expression of the Cap protein of porcine circovirus type 2(PCV2). To examine this hypothesis, we constructed baculovirus vectors that included both Syn21 and P10 UTR, and compared the Cap expression levels relative to Cap vectors lacking the Syn21 and P10 UTR. The expression of Cap in baculovirus-infected cells was then determined by Western blotting and immunofluorescence. The results indicated that Syn21 and P10 UTR could synergistically enhance the expression efficiency of PCV2 Cap. Moreover, the ability of Cap protein to assembled into virus like particles(VLPs) was not affected, and the yield of PCV2 VLPs has been increased by a factor of 4.09 ± 0.04(n=3). In addition, we examined the effect of translational enhancer on transgene expression under the control of CMV promoter,and found Syn21 and P10 UTR could enhance transgene expression. Therefore, the novel baculovirus system has great potential to improve the production of VLPs and subunit animal vaccines.2. Based on the published sequences of TBSV on GenBank, P19 gene was synthesized and then cloned into the baculovirus vector under a constitutive OpIE2 promoter.To check whether P19 is functional in Sf9 cells, we performed a GFP reporter assay wherein the abrogated effect of an established SiRNA against GFP is directly correlated with P19 activity. The results showed that P19 could restore the SiRNA-mediated downregulation of GFP expression up to 89.6 %, whereas the control vector and the mutated form of P19 could not. Therefore, it could be concluded that P19 effectively suppressed siRNA-mediated gene silencing in Sf9 cells. Based on the results above, we further evaluated the effect of P19 expression on baculovirus production and foreign gene expression in Sf9 cells. The results showed that P19 could enhance baculovirus yield, GFP and Firefly luciferase expression levels up to 6.80 ± 0.08, 1.81 ± 0.04, and 2.12 ± 0.02 fold respectively, at 72 h post infection(n = 3). What’s more, it was also proved that transient expression P19 in Sf9 cells could significantly increase the foreign gene expression no matter what time phase of promoters were chosen for P19 expression.3. Baculovirus surface display vector was constructed and then pseudotyped with the membrane anchor ectodomain of vesicular stomatitis virus G protein(VSVG-ED). Four complement inhibitor genes were synthesized and cloned into the novel surface display vector to construct recombinant baculoviruses. The complement inhibitors were effectively expressed on the cell membrane observed with Laser confocal scanning microscopy. To better determine the average extent of vector inactivation in mice and pigs, the sensitivity of baculovirus to serum complement from mice and pigs was examined. The baculoviruses were exposed to normal or heat-inactivated serum from different mice or pigs and then the residual titers were tested. The results showed that baculoviruses have been engineered to display OMCI, pFc and DAF exhibited the survival in mice serum with 39.3%, 65.6% and 90.2%, respectively, whereas the control virus exhibited only 9.4% survival; baculoviruses have been engineered to display pFc, OMCI and SPICE exhibited the survival in mice serum with 75.6%, 42.3% and 36.5%, respectively, whereas the control virus exhibited only 10.2% survival. Thus, the surface-modified baculoviruses exhibited complement resistance, VSVG-ED-DAF showing the highest level of protection in mice serum and VSVG-ED-pFc showing the highest level of protection in pig serum. This novel baculovirus vectors represent a method to overcome the inactivation by the complement that previously has hampered their effective use in animal vaccine applications.4. Based on the previous study, we constructed a novel baculovirus vector vaccine using CSF as an animal disease model and evaluated the immune response. Pig immune experiments indicated that it could induce stronger immune response than the traditional baculovirus vector vaccine. The serum of the pigs produced higher level of CSFV-specific antibodies, neutralization antibodies and IFN-γ, suggesting that this novel baculovirus system has more advantages when it used as animal vaccine vector. The study laid the foundation for a new CSFV vaccine research, and it also provided a new tool and platform for baculovirus-based animal vaccine development. |
