| Porcine parvovirus infection(PPVI)caused by porcine parvovirus(PPV)was responsible for porcine reproductive failure,which caused the predominant clinical signs,such as fetal death,mummification,and stillbirths in pregnant sows,followed by enteritis,respiratory diseases and dermatitis in piglets.Since it was discovered in Germany in 1965,PPVI has resulted in considerable losses to the swine industry throughout the world.The VP2 protein as the major capsid protein of PPV,has the ability to self-assemble into viral-like particles(VLP)in vitro,which containing major neutralizing and hemagglutination sites,and is an attractive target protein for study of PPV epitope,assembly mechanism and subunit vaccine.In this study,liner B-cell epitopes of PPV VP2 protein were identified by peptide scanning,and simultaneously the effects of mutations of critical key amino acids on PPV VP2 VLP assembly were demonstrated using continuous mutation method.Based on previous studies,an efficient and stable PPV VP2 VLP vaccine was developed,and the immunoprotective efficacy was estimated.Moreover,the pilot scale production of PPV VLP vaccine was conducted in the good manufacturing practice(GMP)workshop,and the pilot scale production process as well as the quality of VLP vaccine was evaluated systematically.It mainly includes the following four parts:1.Identification of liner B-cell epitopes of VP2 protein against PPVIn this study,twelve monoclonal antibodies(mAbs)against PPV and PPV VP2 protein were generated by the hybridoma technique,and characteristics as well as epitopes of mAbs were evaluated.Six of mAbs recognized linear B-cell epitopes,and the others may bind with conformational epitopes.Moreover,two conformational mAbs 8E11 and 10A9 were defined to be able to neutralize the standard PPV 7909 strain,with the virus neutralization(VN)titers reaching 1:2,048 and 1:1,024,respectively.Truncation expression and peptide scanning were applied to identify linear B-cell epitopes agaisnt PPV VP2 protein.The peptide,89ESGVAGQMV97 was defined as a dominant linear epitope,and was exposed on the virion surface with the high antigenic index,but weak hydrophilicity.Results from alignment of VP2 protein sequence from different PPV strains indicated that peptide 89ESGVAGQMV97 had a higher amino acid mutation rate at 91G,92V and 93A position.Alanine-scanning mutagenesis further revealed that residues 89E,90S,91G,92V and 94G were the core sites involved in antibody recognition.2.Study on critical key amino acids of PPV VLP assemblyIn this study,in order to identify the key amino acids and intermediate morphology of PPV VLP assembly,ten deletion mutants within the N-terminal of VP2 protein and three site-directed mutations in the full-length of VP2 protein were constructed and expressed using deletion and sitedirected mutagenesis.The structure,function and intermediate morphology of mutant VP2 proteins was evaluated by transmission electron microscopy,dynamic light scattering,sucrose density gradient centrifugation and so on.Our results showed that deletion of the first 47 amino acids from the N-terminal of the VP2 protein did not affect capsid assembly,and further truncation to residue 48 Asparagine(Asn,N)caused detrimental effects.Site-directed mutagenesis experiments demonstrated that N47K reduced the assembly efficiency of PPV VLP,while N48K destroyed the stability,hemagglutination,and self-assembly characteristics of PPV capsids.Sucrose gradient sedimentation experiments indicated that the deletions or mutations of critical amino acid sites could affect or disrupt the formation of intermediates that closely related to PPV VLP assembly.Results from native PAGE further showed that site-directed mutation at N48K did not affect the association of VP2 monomers to form into oligomers,but destroyed the ability of oligomers to assemble into macromolecular particles.3.Evaluatation on immunoprotective efficacy and safety of PPV VLP vaccineIn this study,PPV VP2 protein was expressed by E.coli in high-density fermentation.Purified PPV VP2 protein emulsified with MONTANIDETM ISA 201 VG adjuvant was to prepare PPV VLP vaccine.In the present study,we further employed guinea pigs and pigs to assess the safety,efficacy and immune protection of the PPV VLP vaccine.Immunogenicity assays in guinea pigs and pigs indicated that different doses of the PPV VLP vaccine elicited animals produced higher hemagglutination inhibition(HI)and virus neutralization(VN)antibody against PPV comparable to the commercial inactivated vaccine and negative control(PBS).Lymphocyte proliferation assay indicated that PPV VLP vaccine induced T cell response against PPV in guinea pigs.Results from the virus challenge test showed that when the low dose 2.8μg of PPV VLP inoculating,viral loads in the spleen and liver of challenged guinea pigs were significantly reduced.No significant side effects were observed in safety tests with pigs.Moreover,HI amd VN antibody titers of pigs immunized with VLP vaccine were still above 29,even at 180 dpi.4.Study on the pilot scale production and product quality of PPV VLP vaccineIn this study,five consecutive batch of pilot scale production of PPV VLP vaccine was conducted in the GMP workshop of the cooperating company,Luoyang Huizhong Biotech Co.,Ltd.The compliance and feasibility of production processes were verified.Simultaneously,quality tests for PPV VP2 semi-finished antigen and PPV VLP vaccine was systematically examined in accordance with the requirements of the current Chinese Veterinary Pharmacopoeia.Results from PPV VP2 semi-finished antigen tests showed that the sterility,endotoxin content and HA titers of PPV VP2 antigen meet the requirements.The physical properties and endotoxin content of the VLP vaccine were up to the requirements.Moreover,PPV VLP vaccine were safe,without side effects,and could stimulate guinea pigs produce HI antibody against PPV,with HI titers above 1:64.The quality of PPV VLP vaccine meets the standard for veterinary PPV vaccine.These findings indicated that manufacturing processes for PPV VLP vaccine are feasible and stable,and the PPV VLP vaccine is safe and effective. |
