Developent And Evaluation Of Virus-like Particle Vaccine Against Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2)is the causative agent of porcine circovirus-associated disease(PCVAD),which causes heavy economical losses to globar pig industry.Currently prevalent PCV2 can be divided into types 2a,2b,2c,2d and 2e,four of which are prevalent in China except for type 2c.Presently,the main tool for PCVAD control is vaccination and the available vaccines include inactivated vaccine and recombinant subunit vaccine.The inactivated vaccine is commonly produced in PK-15 cells,which is limited by low titer of PCV2.The subunit vaccine can be expressed in E.coli or baculovirus/insect cell system,which is limited by complex purification and high production cost.In addition,PCV2 vaccine requires suitable adjuvant for efficient stimulation of immune response.However,traditional adjuvants can not stimulate cellular immune response.Novel adjuvants such as montanide ISA206 can stimulate cellular immune response,but its use is limited by higher cost.PCV2 genome contains interferon(IFN)stimulation response element which plays an important role in the viral response to IFN.Previous study showed that the tretment of PK-15 cells with porcine IFN can improve PCV2 production.Elastin-like polypeptide(ELP)is the polymer synthesized using the repeat sequence of elastin with the property of temperature sensitive reverse phase transition.Therefore,ELP fusion proteins can be purified by simple inverse transition cycling(ITC).In addition,ELP has been used as drug delivery vector,tissue engineering material and immune adjuvant due to its low immunogenicity and good histocompatability.Pattern recognition receptors such as Toll-like receptors(TLRs)play important roles in innate and required immune responses and thus TLR ligands have emerged as novel adjuvants.The main aims of this study included generation of PCV2 highly permissive PK-15 cell lines by dilution cloning and IFN gene transfer,prepartion of PCV2 virus-like particle(VLP)vaccine by fusion expression of the Cap protein with ELP,and evaluation of immuoprotective efficacy of VLP vaccine by animal immunization and virus challenge.1.Generation of PK-15 cell lines highly permissive to PCV2 by IFN gene transferIn order to generate PK-15 cell lines highly permissive to PCV2 infection,the parental PK-15 cells were cloneed by dilution cloning and screened by indirect immunofluorescence after PCV2 infection.The result showed that 8.3%cell clones were resistant to PCV2 infection.77.1%or 14.6%cell clones had lower(PK-15lower)or higher susceptibility(PK-15higher).By using Sleepy Beauty(SB)transposon as the gene transfer vector,the fusion gene of porcine IFN-gamma with the immunoglobulin G Fc domain(IFNg-Fc)was transfected into PK-15higher cells and the transgenic cell clones were screened by PCR.Among 96 cell clones screened,42%contained randomly integrated transgene(called PK15-IFNg-FcRan)and the remaining 58%contained SB-integrated IFNg-Fc expression cassette(PK15-IFNgSB).RT-PCR and protein detection showed that the IFNg-Fc fusion protein was correctly expressed in PK15-IFNg-FcRan and PK15-IFNgSB cells with potent antiviral activity.Quantitave PCR showed that the transgene was genetically stable in the two cell lines after 25 passages.Viral titration analysis showed that PCV2 titer(TCID50)was increased by 2.6 or 2.0 log 10 in PK15-IFNg-FcSB or PK15-IFNg-FcRan cell line as compaed with that in the parental PK-15 cells.Quantitative PCR confirmed that PCV2 genome copy numbers in the two cell lines were significantly higher in as compaed with that in the parental PK-15 cells.These data suggest that the two transgenic PK-15 cell lines could be used as IFN production cells and the PK15-IFNgSB cell line without antibiotic resistant gene could be used for PCV2 vaccine production2.Development of ELP-fused PCV2 VLP vaccineTo develop a novel PCV2 subunit vaccine,the coding sequences for the neutralizing epitopes of PCV2a,2d and 2e were fused with PCV2b Cap gene,and the fusion gene was cloned into histidine(His)tag fusion expression vector pET-30a and ELP fusion expression vector pET-ELP,respectively.The recombinant vectors pET-Cap and pELP-Cap were transformed into E.coli BL21(DE3),and the expression of fusion protein was induced by IPTG and purified by nickel affinity column or inverse transition cycling.The results showed that ELP-Cap fusion protein was correctly expressed in E.coli with an expression level of 56.2 mg/L,which was purified to 94.3%with 74.6%recovery and 41.9 mg/L yield after 2.5-h processing.The Cap-His protein had expression level of 49.4 mg/L,which was purified to 90%purity with 60.8%recovery and 30 mg/L yield after 23-h processing.Both ELP-Cap and Cap-His fusion proteins formed typical virus-like particles after purification,named VLP and ELP-VLp,respectively.Mouse immunization study showed that the two VLPs could induce Cap-specific antibody response and stimulate IL-4,IFN-y and TNF-a production.The virus challenge experiment showed that viremic titer of ELP-VLP or VLP-immunized mice was decreased by 2.3 or 1.81og10/ml by day 14 after challenge,as compared with that of unimmunized control group.These data suggest that ELP-VLP was more immunogenic than VLP and could further devloped as a novel PCV2 subunit vaccine.3.Selection of the immune adjuvant for ELP-fused PCV2 VLP vaccineTo select a TLR ligand adjuvant for ELP-VLP vaccine,the lipoprotein Ag473 from Neisseria meningitides(TLR2 ligand),the flagellin FlaB from Vibrio vulnificus(TLR5 ligand)and the heat shock protein 70(Hsp70)from Mycobacterium tuberculosis(TLR2/4 ligand)were expressed in E.coli and the purified proteins,as well as montanide ISA206 and incomplete Freund's adjuvant(IFA)controls,were used to immunize mice with ELP-VLP.The serum samples were collected at days 7 and 28 post immunization for ELISA antibody detection and PBSCs were isolated on day 28 for cytokine detection.Among the three TLR ligands tested,Ag473 had stronger humoral immune adjuvant activity to stimulate antigen-specific antibody response.In addition,Ag473 showed stronger effect to stimulate the production of Thl cytokines such as TNF-a and IFN-y and Th2 cytokines such as IL-4.Although the immunoadjuvant activity of Ag473 is slightly lower than ISA206,it is significantly higher than IFA.Compared to that of the control group,PCV2 viremic titer of ELP-VLP+Ag473-immunized mice was decreased by 3log10/ml.These data suggest that Ag473 is a suitable TLR ligand adjuvant for PCV2 subunit vaccine.4.Evaluation of protective efficacy of ELP-fused PCV2 VLP vaccineThe previous mouse study showed that ELP-VLP was more immunogenic than VLP and lipoprotein Ag473 was a suitable adjuvant for ELP-VLP.In this study,21-day-old piglets were immunized with VLP+Ag473,ELP-VLP+Ag473 or commercial PCV2 subunit vaccine.The serm samples were collected for ELISA and neutralizing antibody detection and PBSCs were isolated for IFN-y detection at different days post immunization.Each immunized piglet was challenged with 105TCID50/ml PCV2 at day 28 post immunization.On days 7,14 and 21 postchallenge,the serum samples were collected for viremic titer detection.The results showed that the ELISA antibody level in ELP-VLP+Ag473 immune serum was lower than that in VLP+Ag473 immune serum,but was similar to that in control vaccine immune serum at day 7 post immunization.ELISA antibody difference between the three immunization groups was decreased from day 14 and became similar on day 28 post immunization.The neutralizing antibosy titers of the three immunization groups were similar from day 14 to 28 post immunization.On day 7 post immunization,the IFN-y concentration in the PBMC culture medium from ELP-VLP+Ag473 immunized pigs was similar to that from VLP+Ag473 immunized pigs,but was significantly higher than that from commercial vaccine immunized pigs.Although their difference was smaller on day 14 post immunization,the IFN-y concentrations of the two VLP immunization groups were still higher than that of commercial vaccine immunization group by day 28 post immunization.Viremic titers of the three vaccine immunization groups were similar,but significantly lower than that of control group from day 7 to 21 postchallenge.These data demonstrated that the immunoprotective efficacy of ELP-VLP was comparable to that of VLP or commercial vaccine.Since much easier and cheaper production,ELP-VLP could be further developed as a novel PCV2 subunit vaccine.