Characterization Of Three Novel Linear Neutralizing B-Cell Epitopes In The Capsid Protein Of Swine Hepatitis E Virus

Hepatitis E virus（HEV）causes liver disease in humans and is thought to be a zoonotic pathogen and has a wide range of hosts in nature.As the first found natural host of HEV,swine is currently considered to be the main host for the transmission of genotype 3 and 4HEV,which share 80-90%homology with human HEV.The genotype 4 swine HEV can be transmitted to humans through food-borne or water-borne pathways.Therefore,it is important for human public health to blocking the transmission of HEV in pigs.HEV ORF2 encodes virus capsid protein,which ensures the integrity of the viral genome and participates in many important biological functions of the virus.HEV ORF2protein contains major immune-dominant antigens and serves a good candidate for the vaccine.Our studies have demonstrated that the genotype 4 swine HEV truncated ORF2protein,sORF2-C（aa 393-660）,has good antigenicity and immunogenicity.Four monoclonal antibodies（MAbs）,three of them,1E4,2C7,and 2G9 were generated by immunizing mice with sORF2-C recombinant protein and one,1B5,was produced and characterized previously,were used in this study.The results showed that three epitopes recognized by 1E4,2C7,and 2G9 were characterized on HEV ORF2 protein by interaction with sORF2-C truncated protein and synthetic peptides.Truncated genotype 4 swine HEV capsid protein（sp239,aa 368 to 606）can form multimeric forms.Mab 2C7,2G9,and 1B5partially blocked the binding of sp239 recombinant protein to HepG 2 cells and swine HEV infection of rabbits.The main works and results are described below:1.Characterization of epitopes on swine hepatitis E virus ORF2 proteinThe binding site of three mAbs 2C7,2G9 and 1E4 on HEV ORF2 were characterized by the interaction with the prokaryotic truncated sORF2-C protein and synthetic peptides.With western blotting or indirect ELISA,and the binding region（4-7 amino acids）was identified as ⁴⁰⁷EPTV⁴¹⁰,⁴⁵⁸PSRPF⁴⁶² and ⁶²⁵DFCP⁶²⁸.With the PyMol software data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein based on the capsid protein crystal structure.The immune capture assay results show that 2C7 coated at a concentration of 200,400,and 800 ng/well,and both 2G9 and 1B5coated at concentrations of 400 and 800 ng/well could capture native swine HEV particles from feces.However,the data suggested that the epitope recognized by MAb 1E4 might not be on the surface.In addition,sequence analysis revealed that the three epitopes recognized by 2C7,2G9 and 1B5 are common to various distinct HEVs isolated from various host species.2.The MAbs neutralized HEV infection in vitro and vivo.Truncated genotype 4 swine HEV capsid protein（sp239,amino acids 368 to 606）can exist in multimeric forms.Preincubation of swine HEV with 2C7,2G9,or 1B5 before addition to HepG 2 cells partially blocked sp239 binding or inhibited swine HEV infection.As the HEV infected animal model,rabbits were used in this study to conduct in vivo MAb neutralization experiments.Swine HEV stock was incubated with each MAb normal mouse IgG,or HEV-infected serum.After challenged,fecal shedding,viremia and antibody level of rabbits in each group were detected with RT-PCR and indirect ELISA.The study indicated that 2C7,2G9,and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG.The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species.3.Evaluation of the synthesis peptides containing the three neutralizing epitopes of Genotype 4 swine HEV ORF2 on the inhibition of swine hepatitis E virus infection.The lack of a highly efficient in vitro cell culture system has hampered traditional HEV vaccine development,genetic engineering of a subunit vaccine is currently being pursued.The results given above suggest that epitopes recognized by MAbs 1B5,2C7,and 2G9partially blocked swine HEV infection of rabbits may generate naturalizing anti-HEV antibodies in vivo after immunization.Therefore,three neutralized epitopes recognized by2G9（EPTV）,1B5（VKLYTS）and 2C7（PSRPF）were used to make the synthetic peptides which were used to immunized rabbits for the protection against swine HEV infection.The study also included the tandem peptides including epitopes of 2G9-1B5（EPTVKLYTS）and2G9-1B5-2C7（EPTVKLYTSPSRPF）.The rabbits were immunized with the indicated peptides,then challenged with swine HEV.The fecal shedding,viremia,antibody and ALT levels were evaluated using RT-PCR and indirect ELISA.The results showed that peptides recognized by 2C7,1B5,and 2G9 can partially protect rabbits against swine HEV infection.The Pep EPTVKLYTSPSRPF and sp239 have better protection against swine HEV infection.