The Preparation And Detection Of Virus-Like Particle-based PCV2b Vaccines

Virus-like particles (VLPs), lacking infectious nucleotides, with immunogenicityof their native viruses and self-assembly by viral capsid protein (CP), have beendeveloped as antigens of subunit vaccines against various viruses. Three human usedvaccines formulated with VLP antigens have been approved for the prevention ofhepatitis B virus (HBV), human papillomavirus (HPV) and hepatitis E virus (HEV).The achievements have dramatically boosted the development of other VLP-basedvaccines. In the case of the p239-based HEV Hecolin vaccine, a robust process wasdeveloped using E.coli as the expression system. However, in the preparation of theVLP vaccine, the CP is in inclusion bodies, after refolding of urea-denatured and thenassembled into VLPs, while the refolding process after treatment by urea has manyfactors difficult to control in industrial production. Further, the three kinds ofcommercial VLP vaccines for human are used for DNA viruses. In animal viruses,there are extensive DNA viruses, among which, porcine circovirus type2and bsubtype (PCV2b) is the major pathogens caused fatal disease in the global pigindustry. In addition, there has been no report of licensed veterinary VLP vaccineworldwide as yet. Therefore, we hope to express CP in soluble form through E. colithen prepare PCV2b VLP vaccine, and on this basis, build the technology platform ofVLP vaccine preparation and detecting.To get the soluble PCV2b-CP-His in E. coli and prepare PCV2b VLP vaccine,we optimized the codons of CP-His encoding gene, synthesized the gene throughmultiple-PCR, and then tried to express the CP-His in E. coli by low-temperaturecultivation; We tried a variety of methods including size exclusion chromatography,ammonium sulfate fractionation precipitation and nickel affinity chromatography toobtain CP-His which would be assembled; To prepare PCV2b VLP, we explored the the assembly condition and assembly buffer solution; The methods of transmissionelectron microscopy and fluorescence spectroscopic analysis were used to monitor theassembly of VLPs; The ELISA and immunization were operated to detect theantigenicity and immunogenicity of VLPs.The major results are:Soluble CP-His with high level was expressed in E. coli.Purifiedly monomeric CP-His was obtained by nickel affinity chromatography.The monomeric CP-His was assembled into homogenous and tight VLPs withdiameter size around20nm.ELISA result showed that VLPs were recognized by the sera from the inactivatedPCV2b immunized mice and the level of recognization was correlated with themorphology of VLPs.In the immunization test, we found that VLP with similar shape to native PCV2bcaused a high level of specific antibodies PCV2b in mice.Fluorescence spectroscopic analysis (FSA) was used for monitoring disassemblyand reassembly of the PCV2b VLPs. The assembled PCV2b VLPs could be detectedas an atypical profile with a peak at320nm while the disassembled VLP only had onewide baseline.The maximum value of emission spectrum was related to the VLPsstate under TEM, and the immunological activity, but there were no significantdifferences in the fluorescence intensity value.In conclusion, CP-His could be expressed in a soluble form in E. coli and thepurified CP-His could be assembled into VLPs which caused a high level of specificantibodies PCV2b in mice. In addition, the FSA could be a useful assay for rapidlymonitoring assembly of VLPs in research and manufacture.