Expression Of The VP1 Protein Of Genotype 3 Duck Hepatitis A Virus In Baculovirus And Assessment Of Its Efficacy In Ducks

Duck viral hepatitis A is an acute,highly lethal infectious disease of ducklings caused by duck hepatitis A virus(DHAV).This disease is one of the most important diseases threating duck industry.In a long period,DHAV of genotype 1(DHAV-1)is dominant in the duck population in China,and since 2013,DHAV of genotype 3(DHAV-3)has been introduced into China and spread rapidly in ducks.At present,DHAV-1 and DHAV-3 co-exist in ducks,and DHAV-3 has become a dominant genotype.Vaccination is an important means for prevention and control of duck hepatitis A.Currently,live attenuated vaccine and inactivated vaccine are two main vaccine types used for duck immunization.However,production of the traditional live vaccines and inactivatedvaccines is highly dependent on chicken embryos,which has many shortcomings such as high cost,the risk of endogenous virus contamination,and production of biological wastes.Of note,rapid progress has been made regarding generation of novel vaccines against DHAV,such as viral vector vaccines,subunit vaccines,virus-like particle vaccines,and oral vaccines.Among these vaccine types,recombinant protein subunit vaccines have the advantages including low cost,high antigen yield and strong immunogenicity,which represent the most potential vaccine type to replace the traditional egg-based vaccines.Therefore,in this study,we aimed to express the protective antigen VP1 of DHAV-3 in baculovirus expression system and to evaluate the immunogenicity of the VP 1-based subunit vaccine in ducks.This study was mainly carried out from the following three aspects:(1)establishment of one-step real-time PCR(RT-qPCR)for DHAV-3 titration and the animal infection model of DHAV-3;(2)preparation of a panel of monoclonal antibodies against the VP1 protein and establishment of sandwich ELISA for quantitation analysis of the VP1 protein;(3)generation of recombinant baculoviruses expressing the VP1 protein and assessment of the immunogenicity of the baculovirus-based inactivated vaccines in ducklings.Thus,the establishment of one-step RT-qPCR,animal challenge model and sandwich ELISA provided technologic fundations for evaluation of the subunit vaccine.In addition,the inactivated vaccines based on the recombinant baculoviruses can induce a rapid antibody response at week 1 post vaccination,and can provide good protection against DHAV-3 challenge in ducklings.Therefore,the recombinant baculoviruses expressing the VP1 protein had a potential to be used for the development of DHAV-3 subunit vaccines.Our results provided a new option for updating of DHAV-3 vaccines and containment of duck hepatitis A.1.Establishment of one-step quantitative real-time PCR and animal infection model for DHAV-3Reliable methods for virus titration and animal infection model are the basis for assessment of vaccine candidates.In the present study,a pair of primer and probe was designed,and a one-step quantitative real-time PCR method based on TagMan probe was established.This method had a high sensitivity,with the detection limit of 250 copy/μL.The one-step quantitative real-time PCR was used to determine virus titers in duck embryos,chicken embryos and cell cultures,as well as to determine virus load in tissues of DHAV-3-infected ducks.Additionally,using a DHAV-3 RY strain isolated in the field,laboratory culture systems of DHAV-3 were set up.Phylogenic analysis based on the VP1 protein showed that the RY strain had a close genetic relationship with the circulating strains in recent years.The virus can replicate at high levels in duck embryos,chicken embryos and chicken embryo fibroblasts.In addition,4-day-old ducklings infected with the RY strain died rapidly within 2 days post infection,and the mortality was 100%.Virus infection caused typical clinical signs and pathological changes of duck hepatitis,including opisthotonus,severe hemorrhage and necrosis in the liver.Histological analysis demonstrated that extensive necrosis of hepatocyte and infiltration of inflammatory cells were observed in the liver.Moreover,high virus loads of the RY strain were detected in the liver,pancreas,kidney and spleen,indicating the virus can replicate in multiple tissues in ducks.Therefore,the establishment of the one-step quantitative real-time PCR for virus titration,laboratory culture systems for virus propagation and animal infection model are the important basis for next-step evaluation of DHAV-3 vaccines.2.Establishment of sandwich ELISA for quantitation of the DHAV-3 VP1 proteinAntigen content of the live attenuated and inactivated DHAV-3 vaccines was determined through measuring virus titers in chicken embryos,whereas there are limited methods used for precise quantitation of antigen content of subunit vaccines.In this part,a panel of monoclonal antibodies(mAbs)against the VP1 protein was prepared,and a sandwich ELISA was established to determine the VP1 yield.BALB/c mice were immunized with the VP1 protein expressed in E.coli,and the mAbs were generated using the hybridoma method.A total of 12 hybridoma cells were screened after a 3-round subcloning,and the ascites were prepared using some hybridoma strains.The results showed that except for the mAb 1D6,the other mAbs exhibited high endpoint titers against the VP 1 protein(>60000),suggesting high affinity of the mAbs to the VP1 protein.Ten out of twelve mAbs can detect DHAV-3 infection in DF1 cells as determined using indirect immunofluorescence assay.In addition,sandwich ELISA was established in which the mAb 2C11 and anti-VP1rabbit polyclonal antibody were used as the capture and detection antibody,respectively.A good linear reactivity with the VP1 protein at different concentrations was observed(R2=0.9993),and the detection limit of this method was 12.5 ng.Therefore,a panel of mAbs against the VP1 protein was prepared,and a sandwich ELISA method with high specificity and sensitivity was established.This method can be used for quantitation of antigen content in the VP 1-based subunit vaccines.3.Expression of the DHAV-3 VP1 protein in baculovirus and assessment of its immunogenicityIn this part,the VP1 protein of DHAV-3 was expressed in baculovirus and the efficacy of the baculovirus-based inactivated vaccines was evaluated in ducklings.Two recombinant baculoviruses were generated based on different strategies.The prototype VP1 gene of DHAV-3 was expressed in rBac-VP1,and a consensus VP1 gene derived from bioinformatics analysis was expressed in rBac-conVP1.Two baculoviruses can replicate to high titers in Sf9 cells,and showed a good genetic stability.In addition,the VP1 protein of DHAV-3 can be expressed efficiently in Sf9 cells.Optimization of the VP1 expression conditions revealed that rBac-VP1 and rBac-conVP1 produced high levels of the VP1 protein in Sf9 cells inoculated with 1 and 5 multiplicity of infection,respectively.The VP1 yield in Sf9 cells inoculated with rBac-VP1 and rBac-conVP1 were 1.8 μg/mL and 0.53μg/mL.The antigens were inactivated with ethyleneimine and emulsified with oil adjuvant to prepare inactivated vaccines.The vaccines induced a rapid antibody response in ducklings at week 1 post vaccination.After DHAV-3 challenge,3 out of 5 un-immunized ducks died,and rBac-VP1 and rBac-conVP1 provided 100%and 80%protection against DHAV-3 challenge.Therefore,our findings suggest that the DHAV-3 VP1 protein expressed in baculovirus is immunogenic and efficacious against DHAV-3.In summary,in the present study,the one-step RT-qPCR for DHAV-3 titration,animal challenge model and sandwich ELISA for VP1 quantitation were established.In addition,two recombinant baculoviruses expressing the DHAV-3 VP1 protein were generated.The inactivated vaccines based on the recombinant baculoviruses can elicit a rapid antibody response and provide good protection from DHAV-3 infection.These baculoviruses may contribute to the development of novel DHAV-3 vaccines and prevention of duck viral hepatitis.