Characterization Of A Neutralizing Monoclonal Antibody Against PRRSV GP5 Protein And Analysis Of The Epitode

Porcine reproductive and respiratory syndrome virus(PRRSV),one of the most economically significant pathogens worldwide,has caused numerous outbreaks during the past 30 years.This impact has become even more significant with the recent emergence of highly pathogenic PRRSV strains from China.Due to the hight rate of mutation,immunosuppression and theantibody-dependent enhancement,commercial vaccines can not provide enough protection and failed to control PRRSV infection.GP5 protein is the major structrual protein of PRRSV,which is glycosylated transmembrain protein.GP5 was considered as a major inducer of netrilization antibody,and a liner neutralizing epitope was identified in early report.But some reports demonstrated that GP5 was poorly immunogenic and not able to induce robust protective responses.In previous research performed in our lab,a monoclonal antibody against GP5 protein of PRRSV-SD16 isolate was generated.This study aimed to validated the neutralization efficacy against PRRSV infection and the characteristic of the epitode Mabs-5D9 recognized.The main works and results of this study were as follows:1.The Isotyping and Purification of Mabs-5D9Mabs-5D9 was classified as Ig M,kappa type through indirect enzyme-linked immunosorbent assay(ELISA)assay.Antibodies were successfully purified from hybirdoma cell culture precipitate by saturated ammonium sulfate and the use of Protrin L Resin.2.The characteristic of the epitode Mabs-5D9 recognizedImmunofluorescence assay(IFA),indirect ELISA assay and Western-Blot assay were performed to evaluate the characteristic of the epitode Mabs-5D9 recognized.The result showed that the epitode was on the ectodomain of GP5,which is comformational and detergent sensitive.Moreover,while the transportation of golgi apparatus was blocked by the treatment of BFA,Mabs-5D9 no longer recognized the epitode.Also,the ability of virion capture of Mabs-5D9 was reduced by deglycosylation treatment,which suggested that the transportationa N-link glycosylation of golgi apparatus is essential for the emergence of this epitode.3.Mabs-5D9 is a broadly netrilizaiton antibodyAfter co-incubation between PRRSV-SD16 and different concentration of Mabs-5D9,the virus was used to inoculate the PAMs.The replication of virus was analyzed with IFA and q PCR assays both at gene and protein levels.The results showed that Mabs-5D9 inhibited PRRSV –SD16 replication in PAMs in dose-dependent manner.Moreover,the progeny virus titers of supernatant ruduced as well.Meanwhile,Mabs-5D9 inhibit another genotype II srtain including GD-HD,JXA1,VR2385,VR2332,Ch1 a,NADC30-Like and genotype I strain GZ11,indicating that Mabs-5D9 is a broadly netrilizaiton antibody.4.The Mabs-5D9 F(ab)2 fragment production and its functionThe Mabs-5D9 F(ab)2 fragment was generated by the use of pepsin digestion.IFA assay was performed to detect the virus-recognition ability of F(ab)2 fragment.The result showed that F(ab)2 can recognize the antigent as intact Mabs-5D9.In conclusion,the present study demonstrated that the Mabs-5D9 act as a boardly neutrilizing antibody,which can inhibit virus replication,including genotype I and genotype II starins.Also,the epitode recognized by Mabs-5D9 is a comformational,detergent sensitive and golgi apparatus transportation-dependent.These findings not only provide new insights into the natuer of neutrilizing antibody and epitode during PRRSV infections,but also provide new strategy of vaccine development and anti-virus treatment.