Analysis Of HEV Neutralization Sites Using Monoclonal Antibodies Directed Against A Virus Capsid Protein

Hepatitis E Virus (HEV) has emerged to be one of the significant pathogens of acute hepatitis, and it often causes epidemic outbreaks. The large epidemic, which occurred in South Xinjiang of China, in 1986~1988, consisted of ~120 000 cases of hepatitis. More than 1 000 patients died in that outbreak. HEV is a non-enveloped RNA virus with an isosahedral capsid which consists of a single structural protein encoded by ORF2. A peptide (NE2) of pORF2 expressed in E.coli was found to naturally form several oligomer forms based on homodimer. The protein was recognized more strongly by convalescent human serum in dimeric form than in monomeric form. The result shows that the significant immuno-predominant epitopes of HEV are present in dimeric form of the protein. Immunization with NE2 protein may prevent experimental infection of rhesus monkeys with HEV. NE2 protein in solutions was characterized in XL-A analytical ultracentrifuge. The predominant component has sedimentation coefficient as 2.65S, molecular weight as 46kDa, friction ratio as 1.69, which is verified dimeric form with medium asymmetry in solutions.Eight monoclonal antibodies (MAbs) raised by NE2 protein were characterized. Thereinto, 6 MAbs react with the homodimers more strongly than the monomers, which suggests that they recognize specific conformational epitopes of HEV. Another 2 MAbs react with the monomers equal to the homodimers, which suggests that they recognize some linear epitopes of HEV. Furthermore, MAb 8C11, 13D8 and 8H3 were found to be able to immune-capture HEV, which shows that they recognize some exposed structure located on the surface of HEV capsid. Cross blocking ELISA tests reveal that epitopes recognized by 8C11 and 13D8 might be located in such close proximity as for the antibodies to inhibit binding of one another in a reciprocal manner., but 8H3 recognizes a distinct epitope. It was noted that the binding between NE2 protein and 8H3 can be enhanced by the pre-binding of 8C11. The equilibrium dissociation constants (KD) of these three MAbs and their Fab fragments were determined by BIAcore biosensor. The KD values of 8C11 and 13D8 were both about 20nmol/L, but the KD values of their Fab fragments were increased by ten folds, presumably because of valence effects. The KD value of 8H3 was 1 000nmol/L. Compared with the native antibody, the KD value of the 8H3 Fab fragment was...