Construction Of Complement Antagonistic Baculoviral Vectors And Their Primary Mechanism For Complement Escape In Vitro

Baculovirus is an enveloped double-stranded DNA virus with numerous advantages as a vaccine vector.For instance,it can not replicate and insert in mammalian cells,thus,possessed good biological safety.Secondly,baculoviral vector can be operated easilyendure high capacity of insertion,transduce plenty of mammalian cells and activate strong natural immunity.Besides,the immunity against baculoviral vector does not existed in mammals.However,baculovirus is highly sensitive to mammalian serum complement,as well as very low transduction efficiency(<0.1%)and low transgenic expression in mammalian immune cells,whichresulted in its limited immune effect.To overcome the complement clearance of baculovirus,multiple genetic and chemical modification methods were faciliated to modify the surface of baculovirus.such as,GP64 envelope protein substitute for VSVG,baculovirus surface modified with PEG,surface display of complement inhibitory protein DAF or MCP,and other strategies to reduce complement clearance of the baculovirus.Our preliminary study found that,(1)the recombinant baculovirus surfacedisplaying porcine IgG1 Fc could evade clearance of complement to virus;(2)Cap protein of porcine circovirus 2(PCV2)can interact with C1 QBP,an important component of classical complement.Thus,we speculated pathway,that Cap might participate in the regulation of complement;(3)Penton protein of Human Adenovirus 3 is an important vehicle,which could self resemble the virus like particles,and also induce a strong humoral and cellular immunity.,We speculated that Penton might involve in r complementregulation to maintain stability and induce adaptive immunity..Based onforementionedresearch,this study aimed to compare the complement escaping efficiency of different recombinant baculoviruses displaying IgG1 Fc?Cap or Penton,to screen the potential component that could protect baculovirus from complement clearance.and toprovide theoretical basis for the construction of efficient baculoviral vectored vaccine for pig.The main results are as follows:1.Construction of baculoviral vector for surface displayingThe porcine IgG1 Fc,Cap protein of PCV2,and Penton protein human adenovirus 3 were inserted into the constructed vector pFBDM-P10-gp64SP-vsvgED,between the gp64SP(N terminal with His-tag)and vsvgTM,then the expression cassette,CAGG-eGFP,wasintroduced as a reporter gene,for evaluation of transduction efficiency in mammalian cells.Finally,four shuttle vectors for recombinant baculoviruses package were constructed,and named pFBDM-P10-gp64SP-vsvgED-CAGG-eGFP,pFBDM-P10-gp64SP-Fc-vsvgEDCAGG-eGFP,pFBDM-P10-gp64SP-Cap-vsvgED-CAGG-eGFP,and pFBDM-P10-gp64SPCap-vsvgED-CAGG-eGFP,respectively.2.The packaging of recombinant baculovirusesAfter transfection with the viral genomes post recombination between forementioned shuttle vectors and bacmids,four recombinant baculoviruses were obtained,namely BV-vsvg-ED,BV-vsvg-ED-pFc,BV-vsvg-ED-Cap and BV-vsvg-ED-penton,which have been identified by PCR,western blot,IFA and laser confocal microscope.3.The efficacy of complement clearance escape after recombinant baculoviruses treated with of swine seraThe results of antagonistic complement ability test in vitro indicated that,escape ability of four recombinant baculoviruses were BV-vsvg-ED-Cap(68.3%),BV-vsvg-ED-pFc(65.5%),BV-vsvg-ED-penton(37.6%)and BV-vsvg-ED(20.1%)in the order from high to low.4.Primary study on the mechanism of recombinant baculovirus escape from complement clearanceThe survival rate of baculoviruses in normal sera was 66.3%,while their survival rate was significantly improved(80.2%)in the sera without IgG and IgM.Thus,we suggested that the classical complement pathwayplay a partial role in baculovirus clearance in swine sera..Moreover,the baculociruses efficacy for complement eacape showed no different no mater the baculociruses replication in insect glycosylated Sf9 cells or mammalian-like glycosylatedmimic Sf9 cellswhich,indicated that the alternative pathway might not involve in baculovirus cleareace in swine sera by complement.complement.Meanwhile,we confirmed that PCV2 Cap protein is an complement antagonist and protectrecombinant baculoviruses escape from complement clearance no mater displaying on viral surface or mix with baculovirus without Cap display.In this study,porcine IgG1 Fc,Cap of PCV2,Penton of Adenovirus 3 were displayed on the surface of baculovirus,to screen the potential complementantagonist which could protect baculovirus escape from complement clearance in swine sera..The results demonstated that porcine IgG1 Fc and PCV2 Cap could be good component to protect baculovirus escape from complement clearance.These studies accelerate the process of application of bculoviral vector as vehicle for the swine vaccine.