Establishment And Application Of Neutralizing Models Against Hepatitis E Virus

Hepatitis E(HE)is a viral hepatitis caused by hepatitis E virus(HEV).In recent years,HEV has gradually became one of the most important causes of acute viral hepatitis in the world.Pregnant women and the elderly are the main high-risk groups.The mortality rate ranges from 0.2%to 4%among the hepatitis E cases.However,pregnant women with HEV infection may have a case-fatality rate of 10-25%.HEV is a non-enveloped virus with a single-stranded RNA genome.The 7.2-kb viral genome contains three open reading frames(ORFs),of which ORF2 exclusively encodes a major viral capsid protein 660 amino acids in length that is responsible for virion assembly and attachment to host cells.p239 which is expressed from a.a.368-606 of HEV pORF2 can self-assemble into virus-like particles(VLP).p239 presents the immune-dominant neutralization epitopes as native HEV particles and can be used for HEV early entry study.A method for evaluating neutralization is needed to assess an effective immune response of host against the virus after HEV infection.The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay(IFA).The neutralizing assay based on real-time PCR calculates the quantities of virus by detecting RNA,which are highly sensitive but operationally complicated and poorly repeatable.IFA ensures that neutralization post-attachment can be tested.However,it is time-consuming and lack of viral source.In this study,we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 and staining with allophycocyanin-conjugated streptavidin(streptavidin APC)using flow cytometry.Using this method,we quantitatively evaluated the neutralization of HEV-specific antibodies and sera.The neutralization assay based on p239 was used to evaluate adsorption blocking.However,neutralization post-attachment can't be tested.Therefore,a neutralization model based on HEV was established to evaluate neutralization of antibodies against viral adsorption,entry and replication.Tradition IFA neutralizing assay was insensitive because of inefficient replication of virus,resulting in fluctuation of the assays.We firstly took advantage of highly replicated HEV genotype 3 Kernow to establish a novel neutralizing assay.A linear regression analysis indicated that there was a high degree of correlation between IFA and the VLP assay.Using the IFA neutralizing assay,we also proved that the major neutralizing antibodies present in sera recognized determinants on p239.Therefore,we were able to quantitatively evaluate the neutralization of HEV antibodies and sera using the VLP neutralization model.The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera using VLP neutralizing model.It indicated that IgG represented the major neutralizing antibodies in sera.However,the neutralization titres of the sera were also influenced by anti-HEV IgM responses.Further analysis also indicated that,the proportions of neutralizing antibodies in the infected macaques' sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels.Thus,the infection more efficiently stimulated neutralizing antibody responses.While evaluating the neutralization of HEV-specific antibodies against Kernow,we found that Kernow couldn't be neutralized with 8G12.It was caused by virus mutation at the site of 554.Furthermore,virus could escape from recognizing of immune system via virus mutation of this site,which might be risk in the population.In conclusion,a novel,high-throughput neutralizing assay based on VLP was developed in this study.Using this method,we could evaluate neutralization of HEV-specific antibodies and sera,which provided a new detection method for HEV diagnosis.However,it could only test adsorption blocking of antibodies.IFA based on Kernow was used to ensure that neutralization post-attachment could be tested.But it was not adapted to high-throughput experiment for time-consuming and low virus production.These two neutralization assays have complementary advantages and will be useful in HEV research.