Analysis of hepatitis E virus neutralization sites using monoclonal antibodies directed against a virus capsid protein.

The dimeric form of the recombinant peptide (E2), comprising amino acid 394-606 of the capsid protein of hepatitis E virus (HEV), is strongly recognized by HEV reactive human serum, and when used as a vaccine, it protects rhesus monkeys against experimental HEV infection. In this work, the relationship of E2 to HEV has been probed using three murine monoclonal antibodies, 8C11, 13D8 and 8H3, all of which react predominantly against the E2 dimer, and can effect immune capture of the virus as well. 8C11 and 8H3 were further found to neutralize HEV infectivity in animals. Cross-blocking patterns between these antibodies discerned two spatially separate antigenic domains, one identified by 8C11 and 13D8, and the other, by 8H3. Kinetic studies using BIAcore biosensor suggest that the epitope to which 8H3 is directed is partially masked, and thus has limited access by the native antibody. However, this is not the case with the smaller Fab. Access to the 8H3 epitope was enhanced by the binding of 8C11, and inhibited by the binding of 13D8 to a distal site on the peptide. Similar to the effects of binding 8H3 to E2, 8C11 was found to enhance immune capture by 8H3, while 13D8 was inhibitory. Moreover, 8C11 and 8H3 act synergistically to neutralize HEV infectivity. The parallel cross-reaction patterns that these antibodies exhibit against the peptide and the virus, respectively, implicate two interacting conformationally dependent neutralization sites on the HEV particle. These sites might cooperate in the adsorption and penetration of the HEV virus.

A bacterially expressed particulate hepatitis E vaccine: antigenicity, immunogenicity and protectivity on primates.

It was evaluated its antigenicity, immunogenicity and efficacy of a candidate recombinant hepatitis E virus (HEV) vaccine, referred hitherto as HEV 239 vaccine. The vaccine peptide has a 26 amino acids extension from the N terminal of another peptide, E2, of the HEV capsid protein, which has been shown to protect monkeys against HEV infection previously. The vaccine peptide is similar as E2 in that: first, the vaccine peptide migrates predominantly as dimer in SDS-PAGE and it is dissociated into monomers by heating; second, its dimeric form of which predominantly recognized by HEV reactive human serum; and third, it shows the same pattern of reaction as E2 with a panel of eight monoclonal antibodies that had been raised against E2. In contrast to E2, the vaccine peptide aggregates to form particles of 13 nm mean radius, and consequently, it is more than 240 times more immunogenic than E2. Using alum as adjuvant, immunizing dose determined in mice was 80-250 ng for the vaccine and >60 microg for E2. Rhesus monkeys twice vaccinated with a 10 microg or a 20 microg formulation of this vaccine showed essentially the same antibody response, whereas the response to a 5 microg formulation was delayed but reached similar antibody levels. All the three vaccine formulations afford complete protection against infection with 10(4) genome equivalent dose of the homologous genotype 1 virus. At higher virus dose of 10(7), the same vaccine formulation partially protected against the infection and completely protected against hepatitis. The efficacy of the vaccine was essentially the same for the homologous genotype 1 virus and heterologous genotype 4 virus.

Evaluation of antibody-based and nucleic acid-based assays for diagnosis of hepatitis E virus infection in a rhesus monkey model.

We have evaluated four hepatitis E virus (HEV) specific antibody assays, using sequential samples taken from 86 rhesus monkeys at intervals for up to 86 weeks after they had been infected with different doses of HEV. The animals are a common experimental model of hepatitis E. The large collection of sequential samples used avoids uncertainties encountered in previous studies regarding the precise infection status of study subjects and minimizes bias due to the individuality of response to infection. One assay (YES IgG) was produced with synthetic peptides; the others (E2 IgM, E2 IgG, and GL IgG) were produced with recombinant antigens. The results were compared with the viral RNA contents of the serum and stool samples and the occurrence of these virological and immunological markers in the course of the infection was temporally related to the development of hepatitis. Diagnostic utility of the markers was assessed according to their response rates and prevalence at different times in the course of infection. All the animals produced E2 IgG and developed viremia and all but one also produced E2 IgM and excreted the virus in stool, whereas response rates for the other antibodies were lower and decreased with virus dose. Hepatitis occurred over a period of 4 weeks between 3 and 7 weeks after infection. Virological activity occurred mainly during the incubation period and the prevalence of viral markers declined rapidly after the onset of hepatitis. Production of the E2 antibodies immediately preceded the onset of hepatitis, and this was followed about one week later by production of the other antibodies. Seroprevalence E2 IgM reached a peak value 3 weeks after the onset of hepatitis, whereas seroprevalence of GL IgG and YES IgG peaked after the disease had subsided. E2 IgG persisted in all animals for the entire duration of the experiment of up to 86 weeks and possibly beyond and, thus, can serve as a useful epidemiological marker of HEV infection.