Study Of PCV2 Genetic Engineering Vaccine

Porcine circovirus type 2 (PCV2) could cause PMWS (Post-weaning pigsmultisystemic wasting syndrome), PDNS (porcine dermatitis and nephropathy syndrome),PNP (proliferative and necrotizing pneumonia), CT (congenital tremors), PRDC (porcinerespiratory disease complex) and reproductive disturbance. PMWS was first reported in1990s in Canada. From then on, PCV2 had widely spread in the world and maintain highinfection rate in herd. PCV2 could cause progressive weight lose and high wasting, on theother hand the virus proliferates in the immune system of piglet, and cause disorder ofimmune function. The virus has caused great economic lose in the pig industry. PCV2was first found in 2000 in our country, and now the infection rate of PCV2 in our countryis almost 50ï¼…. Vaccine inoculation is the main method to prevent and control this disease.But low virus yield in cell make it difficult to develop traditional vaccine such as inactiveand active vaccine. It has become a focus to develop safer and more effective and cheapvaccines to prevent and control PMWS. However, due to limited immunity in severalexperimental vaccines, the progress of new vaccines against PCV2 maintains slowly.Therefore, in the present study, we explored the new vaccine design against PCV2, andconstructed subunit vaccine based on baculovirus expression system, modified ORF2DNA vaccine and the recombinant viruses based on baculovirus expression system.Furthermore, the immune efficacy of the candidate vaccines was investigated. The mostresearch works were as following:1. Construction and immune effect of PCV2 subunit vaccineTo construct the transfer plasmid, open reading frame (ORF) 2 of PCV2Yu-A strainwas amplified by PCR. ORF2 sequence was cloned into the corresponding sites ofbaculovirus transfer vector pFastBacTM (Invitrogen). Transfer vector was transformedinto E. coli DH10Bac (Gibco) containing baculovirus shuttle vector (bacmid) and helpervector. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. Thecolonies of E.coli containing recombinant bacmid (rBac.ORF2) were collected threetimes by blue/white selection. After isolation and purification, rBac.ORF2 DNA wastransfected into sf9 cells to yield recombinant baculovirus Ac.ORF2. Expressed ORF2protein was confirmed with indirect immunofluorescent assay and western blotting usingrabbit anti-PCV2 polyclonal antibodies. When the recombinant ORF2 protein in insectcells was partially purified by gradient centrifugation, some virus-like particles (VLPs)with morphology similar to PCV2 virus were detected by negative staining electronmicroscopy. The diameter of the particles was estimated to be of the order of 17 nm. Some of the observed particles had a dark-stained centre that implied that they wereempty capsids. To further study the immunogenicity of the VLPs, PCV2-free piglets werevaccinated with the crude lysate from recombinant baculovirus (Ac-ORF2)-infectedinsect cells, at doses of 0.1 mL (10⁶ cells), 0.5 mL (5×10⁶ cells) or 1.0 mL (10⁷ cells).PCV2 antibodies can be definitively detected at 4 weeks in pigs following a primingimmunization with 0.5 mL or 1.0 mL of crude lysate from Ac-ORF2, and the levelincreased steadily after a boost immunization. However, there was no significantdifference between these groups. No detectable PCV2 antibody response was induced inthe group immunized with 0.1 ml of crude lysate before or after the boost. Except for therelatively high humoral immune response obtained in this study, the higher PCV2 specificlymphocyte proliferation responses were developed in piglets immunized with 0.5 mL or1.0 mL of crude lysate, especially with 1.0 mL. Thus, the expressed ORF2 protein hassignificant potential as a subunit vaccine against PCV2 infection.2. Construction and immune effect of modified DNA vaccines against PCV2Studies have demonstrated that directing antigen to different subcellularcompartments can affect the processing and presentation of the antigen, which, in turn,can alter the character of the immune response to the antigen. Whether thenuclear-targeted PCV Cap protein affects antigen presentation to the host immune systemand consequently influences the elicited immune response? To elucidate this question, inthe present work, we constructed four different expression plasmids encoding Cap proteinwith different subcellular localization: cytoplasmic (Cy-ORF2), secreted (Sc-ORF2),membrane-anchored (M-ORF2) or nuclear (pc-ORF2). Indirect immunofluorescent assaydemonstrated that the modified ORF2 gene could be expressed and the expressed proteinswere targeted into expected cell compartments of the transfected cells. Using mousemodel, the immunogenicity of the modified Cap proteins was characterized by DNAimmunization. The data obtained in the present study clearly demonstrates that targetingthe PCV2 ORF2 protein to different subcellular compartments does indeed influence boththe humoral and cellular immune responses that are elicited by i.m. DNA vaccination.Following immunization, although all four DNA expression constructs could inducePCV2-specific humoral immune responses, mice inoculated with Sc-ORF2 or M-ORF2developed significantly high level of neutralizing antibodies than those received pc-ORF2or Cy-ORF2. More importantly, compared to other DNA vaccine constructs, enhancedcellular immune responses could be elicited in mice immunization with M-ORF2, asdemonstrated by improved T-cell proliferative activities and IFN-γlevel. Second was themice immunized with native ORF2. Relatively lower level of cell-mediated immune responses was observed in mice immunized with Sc-ORF2 and Cy-ORF2. Therefore,taken the induced humor and cell-mediated immune responses into consideration,membrane-anchored (M-ORF2) shows the best immunogenicity and may be used as astrategy to develop a new genetation of vaccine against PCV2 in the future.3. Construction and immune effect of recombinant baculovirus expressing the PCV2ORE2 proteinAlthough AcMNPV are failing to replicate in vertebrate cells, it does express alinegenes that are dependent on the strength of the promoter used to drive transcription of theforeign gene. Follwing these findings, baculovirus have emerged as a vector with greatpotential for gene transfer in mammalian cells. However, in vivo gene delivery bysystemic administration is hindered by the vector inactivation mediated by thecomplement system. Therefore, in this study we describes the generation of a recombinantbaculovirus in which the vesicular stomatitis virus glycoprotein G (VSV-G) is present inthe viral envelope. The gene encoding VSV-G was inserted into the baculovirus genomeunder the control of the polyhedrin promoter such that it was expressed at very high levelsin infected insect cells but not in mammalian cells. This protein was demonstrated toenhance the escape of baculovirus vectors from intracellular endosomes, increasing thetransduction efficiency of the virus. Two viruses were constructed to carry either thePCV2 ORF2 gene or EGFP gene under the control of the cytomegalovirusimmediate-early promoter-enhancer and express vesicular stomatitis virus glycoprotein(VSV-G) in the viral envelope by inserting the VSV-G coding sequence downstream ofthe polyherdrin promoter. The greater gene transduction efficiency of the Ac-V-EGFP wasconfirmed by comparing the EGFP expression level in a variety of multiplity ofinfection.To characterize the induction of antigen-specific immune response mediated bybaculovirus, mice were subjected to intramuscular with different doses of baculovirusvectors ranging from 10⁸ to 10¹⁰pfu/mL in a 100μL volume. ELISA analysis showed thatrelativatly hifher PCV2-specific antiboby was detected in mice immunized with10¹⁰pfu/mL of Ac-V-ORF2 after the primary immunization. Following a boost at week 3,antibodies increased significantly and gradually increased in mice vaccinated withAc-V-ORF210¹⁰pfu/mL, the mean ELISA antibody level was reach to 1:7169 and theneutralizing antibody titer was up to 1:16. In agreement with the humoral immuneresponse, at 6 weeks alter primary immunization, the highest celluar immune responsewas found in restimulated splenocytes from mice immunized withAc-V-ORF210¹⁰pfu/mL. Taken altogether, Ac-V-ORF2 with the dose of 10¹⁰pfu/mLshows the best immunogenicity and may be used as a strategy to develop a new genetation of vaccine against PCV2 in the future.