Preparation And Immunological Efficacy Of PCV2b Virus-like Particles Embedded With IBDV B Cell Epitopes

Infectious bursal disease virus(IBDV)is the causative agent of a highly contagious disease(IBD)that threatens the poultry industry.It can cause high mortality in young chickens.Until now,no researches have been reported on chimeric VLP(Bepi-Cap-VLP)vaccine in which porcine circovirus 2b(PCV2b)capsid protein(Cap)presents IBDV B-cell epitopes(IBDV-Bepi).Thus,we designed a chimeric protein(Bepi-Cap)in which the PCV2b Cap was exploited as the scaffold for fusing IBDV-Bepi.Bepi-Cap was expressed in E.coli,purified and assembled into Bepi-Cap-VLP.Immune efficacy of the Bepi-Cap-VLP was also detected.The results were as follows:1.B-cell epitope prediction of IBDV capsid protein(IBDV-Cap)To predict B-cell epitopes of IBDV-Cap,we used SWISS MODEL website,Protean software and BepiPred 1.0 website to mimic IBDV-Cap structure and predict candidate peptides.SWISS MODEL(https://swissmodel.expasy.org/interactive),a server for protein homology modelling,was utilized to generate the quaternary structure of IBDV-Cap.Potential B-cell epitopes(210-217aa,242-247aa,274-280aa,309-317aa)were predicted with this model;By analyzing the secondary structure of IBDV-Cap,seven potential peptides(20-35aa,71-77aa,191-199aa,203-209aa,311-317aa,395-401aa,410-417aa)harboring B-cell epitopes were predicted in Protean software.In addition,we also utilized BepiPred 1.0(http://www.cbs.dtu.dk/services/BepiPred-1.0/),a web server,to predict B-cell epitopes and have screened two linear peptides(14-26aa,307-322aa).Based on the prediction,we selected 13 candidate peptides for further analyzing their consensus sequences and obtained the sequence of 3 putative peptides,from which the sequence of a peptide was finalized to be used as IBDV-Bepi for constructing Bepi-Cap-VLP.2.Selection of peptide from PCV2b Cap to be replaced by IBDV-Bepi.With the reference of the previous work,we located three neutralizing epitopes(69-83aa,113-131aa,193-207aa)in PCV2b Cap for being replaced with the IBDV-Bepi.Each of the peptides in the PCV2b Cap was putatively replaced with the IBDV-Bepi to generate three mimic proteins(Cap-Epi?A,Cap-Epi?B,Cap-Epi?D),respectively.Using the SWISS MODEL,we compared the tertiary structure of Cap-Epi?A,Cap-Epi?B,Cap-Epi?D with the PCV2b Cap and found that the structure of Cap-Epi?A was highly similar to that of PCV2b Cap.The surface-exposed IBDV-Bepi could be easier accessible in Cap-Epi?A than that in Cap-Epi?B and Cap-Epi?D.Thus,the epitope(69-83aa)located in Cap-Epi?A could be replaced by IBDV-Bepi.3.Expression and purification of Bepi-CapUsing the plasmid(pET28a-D-His)expressing the PCV2b Cap as a template,an encoding gene expressing the Bepi-Cap was synthesized by overlapping extension PCR and cloned into a pET28a plasmid(pET28a-BepiCap).pET28a-BepiCap was transformed into BL21(DE3)for constructing engineered bacteria(BL21-Bepi-Cap).BL21-Bepi-Cap was cultured at 37? in a shaker and the Bepi-Cap were expressed under IPTG induction(1mM).SDS-PAGE showed that high yields of soluble Bepi-Cap were expressed,accounting for almost 20%relative to proteins from the entire cell lysate.Furthermore,the Bepi-Cap could be obtained by ammonium sulfate precipitation in high purity(>95%).4.Assembly of Bepi-CapThrough in vitro dialysis,the Bepi-Cap were assembled into VLPs(Bepi-Cap-VLP)with around 18 nm,resembling the natural structure of PCV2b.5.Immune efficacy of Bepi-Cap-VLPTo validate whether the IBDV-Bepi was exposed on the surface of Bepi-Cap-VLP,the mice immunized with Bepi-Cap-VLP was prepared to raise antibody for detecting IBDV in dot blot assay.The results showed that antibodies could recognize IBDV.It confirmed that the IBDV-Bepi was surface-exposed on the Bepi-Cap-VLP.To detect whether the Bepi-Cap-VLP could efficiently induce antibodies,we immunized mice with Bepi-Cap-VLP,inactivated IBDV,IBDV-Bepi tandem polypeptide(4T-7B)and PBS,respectively and detected the levels of antibodies in the sera collected at different time points by Elisa.The results showed that:1)The mice immunized with Bepi-Cap-VLP induced higher level of antibodies against 4T-7B than that the mice immunized with inactivated IBDV.2)The mice immunized with Bepi-Cap-VLP could elicit antibodies against inactivated IBDV.To explore the effects of Bepi-Cap-VLP on the immune cells in vivo,we detected the percentages of CD19~+B cells,CD4~+T cells and CD8~+T cells in the lymph nodes from the mice stimulated 48h after boosting immunization on the 63rd day of primary inoculation.Moreover,to further explore the function of heterologous scaffold,the number of bursal cells stimulated by Bepi-Cap-VLP or inactivated IBDV was counted.The results showed that the Bepi-Cap-VLP could promote the proliferation of bursal cells.It indicates that the PCV2b Cap scaffold may enhance B cells proliferation in the bursa of Fabricius.In conclusion,using the PCV2b Cap as the scaffold,the chimeric VLP presenting IBDV-Bepi could be constructed and assembled to elicit immunity against IBDV.The study provides a reference for building chimeric VLP platforms in the prokaryotic system.