IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein

Abstract

To develop an enzyme immunoassay (EIA) for IgM antibody to hepatitis E virus (HEV) (IgM anti-HEV) and IgG antibody to HEV (IgG anti-HEV), a synthetic gene encoding several liner immunodominant antigenic epitopes from HEV structural proteins was assembled as a chimeric recombinant mosaic protein (Mpr) with glutathione S-transferase and used as an immunodiagnostic target. In addition, a neutralization confirmation test was developed using individual synthetic peptides. Among 614 patients with acute hepatitis from 10 geographically distinct outbreaks, IgG anti-HEV was found in 546 (88.9%), with a range of 77-100% depending on the outbreak. Of 130 patients tested for IgM anti-HEV, 126 (96.9%) were positive. Among patients tested within 4 months of onset of jaundice, 37/37 (100%) were IgG anti-HEV positive. For patients from whom sera were collected 1-16 days after onset of jaundice, the geometric mean IgG titer (GMT) was 1:47,000; the GMT increased to 1:70,710 30-40 days after onset of jaundice and decreased to 1:1,778 3-4 months after the onset of jaundice. For patients tested 6-8 months after onset of jaundice, 11/12 (92%) were IgG anti-HEV positive, and the GMT was 1:2,908. IgM anti-HEV was detected in 43/43 (100%) sera collected 1-40 days after onset of jaundice, and the GMT for IgM anti-HEV was 1:10,000 at that time. For sera collected 3-4 and 6-12 months after onset of jaundice, 7/14 (50%) and 5/12 (40%) respectively, were IgM anti-HEV positive. In conclusion, an artificial mosaic protein composed of linear antigenic epitopes from open reading frame 2 (ORF2) and ORF3 of HEV has been successfully applied to the development of a sensitive and specific EIA for the detection of IgG and IgM anti-HEV activity. These assays were used for the verification of HEV infection in outbreak settings and for the diagnosis of HEV infection in sporadic cases.

Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993

“…Therefore, we speculate that anti-HEV IgG antibodies persist for more than 10 years after the onset of sporadic acute hepatitis E in industrialized countries with low endemicity as well. Regarding the duration of seropositivity for anti-HEV IgM, it has been reported that, of sera collected from patients during various hepatitis E outbreaks 3-4 and 6-12 months after the onset of jaundice, 50 and 40 %, respectively, were positive for anti-HEV IgM antibodies (Favorov et al, 1996). Our patient continued to be positive for anti-HEV IgM antibodies for more than 9 months during the convalescent phase.…”

Detection of Sporadic Cases of Hepatitis E Virus (HEV) Infection in China Using Immunoassays Based on Recombinant Open Reading Frame 2 and 3 Polypeptides from HEV Genotype 4

“…Anti-HEV IgM and IgA have been detected in the acute phase of hepatitis E and may disappear during the convalescence period, so that diagnosis of hepatitis E may be improved by detecting anti-HEV IgM and IgA antibodies also (6,8,9,12). …”

“…The following recombinant plasmids were derived from HEV T1 (genotype 4 [30]) and were cloned in pGEM-T (Promega, Madison, Wis.): R11, which contains 902 bp from nucleotides (nt) 4627 to 5529; E4, which contains 725 bp from nt 5343 to 6067; F2-2, which contains 521 bp from nt 6006 to 6526; and E5, which contains 781 bp from nt 6384 to the 3Ј end (nt 7164). It should be noted that a single nucleotide insertion in genotype 4 HEV potentially results in an additional 12 residues at the amino terminus of the ORF2 protein and the loss of 10 residues from the amino terminus of the ORF3 protein (12).…”

Identification of Immunodominant and Conformational Epitopes in the Capsid Protein of Hepatitis E Virus by Using Monoclonal Antibodies

“…More recently, the use of larger overlapping peptides has revealed some conformational epitopes which are reactive with acutephase 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 particles, or indeed whether this antibody repertoire is maintained during the convalescent phase after HEV infection and thus contributes to humoral immunity.…”