ARTIFICIAL MOSAIC PROTEINS AS NEW IMMUNODIAGNOSTIC REAGENTS - THE HEPATITIS-E VIRUS EXPERIENCE

Background: Naturally occurring viral proteins derived from cell cultu re and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies. However, immunoassays utiliz ing these proteins often yield false-positive reactions suggesting tha t it may be possible to identify and remove regions responsible for th ese non-specific reactions. Objective: In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity.Study design: A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short ol igonucleotides by the polymerase chain reaction (PCR). The polypeptide comprises a mosaic of three antigenically dominant regions from the p rotein encoded by open reading frame 2 (ORF2), one antigenically activ e region from the protein encoded by ORF3 of the Burmese HEV strain, a nd one antigenically active region from the protein encoded by ORF3 of the Mexican strain. The mosaic protein was expressed in Escherichia c oli as a chimera with glutathione-S-transferase or beta-galactosidase. Results: Guinea pig sera containing antibodies to the corresponding H EV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein. Both hybrid proteins were shown by immunoblot analysis using a panel of hu man anti-HEV-positive and -negative sera to be HEV-specific. A sensiti ve and specific EIA was developed to detect IgG anti-HEV activity in h uman sera. A neutralization test using individual synthetic peptides c orresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA. Conclusion: An artificial mosaic protein comp osed of short linear HEV-specific antigenic epitopes was constructed f rom synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera.