Consistent and transient antibody responses to hepatitis E virus detected by Western immunoblot using open reading frame 2 and 3 and glutathione em S /em -transferase fusion proteins. other in ORF2.1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may Tos-PEG4-NH-Boc be important in immunity to HEV infection. Hepatitis E virus (HEV) is responsible for epidemic and sporadic cases of enterically transmitted viral hepatitis, particularly in the developing world (17, 31). HEV is a single-stranded, positive-sense RNA virus, with the genome encoding three open reading frames (ORFs), of which ORF2 Tos-PEG4-NH-Boc encodes the major structural or capsid protein, PORF2. Antibody is sufficient to confer immunity to HEV infection (38), but little is known of the structure of the viral particle or of the antibody specificities which contribute to humoral immunity, which could pose a major hurdle in the development and clinical evaluation of effective vaccines. The use of peptide scanning has led to the identification of a number of linear epitopes within the capsid protein of HEV, with many of these being type specific (4, 12, 13, 15). More recently, the use of Tos-PEG4-NH-Boc larger overlapping peptides has revealed some conformational epitopes which are reactive with acute-phase sera (16). Linkage of a number of such peptide epitopes from different strains of HEV into an artificial mosaic protein improves the detection of acute-phase HEV antibody (5, 14), but the antibodies induced with this protein do not appear to be neutralizing in a cell culture system which measures virus-cell binding (26). In addition, it is not known whether antibodies to any of these linear and conformational peptide epitopes can bind to intact viral FHF4 particles, or indeed whether this antibody repertoire is maintained during the convalescent phase after HEV infection and thus contributes to humoral immunity. Expression of PORF2 with an N-terminal truncation of 111 amino acids (aa) in the baculovirus system results in the production of virus-like particles (VLPs), which, in contrast to synthetic peptides or full-length PORF2, appear to mimic the antigenicity and immunogenicity of the native virus (24, 25, 32). Most significantly, immunization of macaques with VLPs confers immunity to both homologous and heterologous virus challenge (37, 38, 43). The improved antibody reactivity and immunogenicity of VLPs have been attributed to conformational epitopes which are not presented by synthetic peptides, full-length PORF2, and most HEV proteins expressed in (24, 25), but the relevant epitopes have not been identified. Expression of the ORF2.1 fragment of PORF2 (aa 394 to 660) in also results in the presentation of conformational epitopes (2, 22, 23). Since antisera from animals immunized with ORF2.1 are able to inhibit the reactivity of HEV patient sera against VLPs by as much as 97% (21), it appears likely that the major epitopes within VLPs and the ORF2.1 antigen expressed in may be the same or closely overlapping. In this study we have used monoclonal antibodies (MAbs) to study the antigenic structure of HEV in more detail. Tos-PEG4-NH-Boc We show that the conformational ORF2.1 epitope involving aa residues.