Research Of Functional Regions On HEV Capsid Protein E2s Domain

Hepatitis E virus(HEV)is the leading cause of Hepatitis E(HE)which is one of the major acute viral hepatitis worldwide.HEV genomic coding region consists of three Open Reading Frames(ORFs),while ORF2 encodes the capsid protein,which is the major component of the outer layer of HEV.The capsid protein palys an important role in host recognization,infection,pathopoiesis and host immune response inducing,etc.Recent studies proved that the homologous dimer formed by HEV a.a.459-606(E2s domain)induced the major neutralizing of HEV,and it was the smallest unit of HEV immunodominance.Further researchs showed that the domains which different neutralizing antibodies recognized were independent.It suggested that several independent epitopes were presented on E2s domain.However,the roles of these epitopes in mechanism of immunization and neutralization of HEV is still unclear.In this research,we developped a method to cluster epitopes of HEV capsid protein with monoclonal antibody(mAb)panel analysis and researched on the corresponding functions of each cluster.Firstly,mAbs,whose binding sites exposed sufficiently on the surface of HEV capsid,were clustered based on their cross blocking datas.Secondly,the amino acids on the surface of HEV capsid protein were mutated by alanine scanning to identify the mAbs in different groups recognized,the sensitive amino acids of epitopes were also located.Then epitopes presented on the E2s domain were determined by the key recognization sites of different mAbs.Finally,the neutralization activity and immunodominance of mAbs in different groups were detected and neutralizing epitopes and immunodominant epitopes on E2s domain were identified based on cluster cases.Six independent epitopes with different properties were also identified in this reseach:C1 was extended from a.a.606 nearby the interface of the dimer to a.a.525 close to HEV P1 domain and a.a.530 in C6,antibodies in C1 had no neutralizing activity and were not immunodominant;C2,with a.a.549 and a.a.591 as the key binding sites,involved neutralizing epitopes but not immunodominant;C3,with a.a.562 and a.a.585 as the core sites,located on the top of dimer and extended to a.a.532 close to C1.Antibodies recognizing C3 could react to genotype 3/4 HEV but not genotype 1 HEV,and had weak neutralizing activity;C4 mainly covered a.a.488-490 and extended to a.a.532,antibodies recognizing C4 were also specific to genotype 3/4 HEV,and had weak neutralizing activity;C5 covered amino aicd sites include a.a.479,a.a.496,a.a.512,a.a.534,a.a.573-578,etc.Antibodies recognizing this group,8C11 as a representative,had strong neutralizing activity and take part in HEV immunization;C6 had overlaps to C5 but was closer to the interface and top of dimer,key binding sites of this C5 is a.a.534 and a.a.535,it was neutralizing epitopes and also took part in HEV immunization.In summary,according to characteristic analysis and statistical cluster analysis for mAbs,we found that six independent epitopes presented on the surface of HEV E2s domain.The recognizing sites of anti-HEV antibodies mainly located on the side face of the monomer in E2s dimer,while antibodies recognized the top of E2s domain also had weak neutralizing activity.Host immune responses to HEV were probably induced by joint actions of multiple epitopes.Among six epitopes identified in this research,C2,C5 and C6 were the mainly recognization domains in host immune responses and the mainly neutralization domains.Our research could contribute to explain the probable molecular mechanisms of immunization,netralization of HEV and the protecting effects of HEV vaccine.The method developped in this study also offers a guide of functional domain research on capsid protein for other viruses.