Antiserum generated by DNA vaccine binds to hepatitis E virus (HEV) as determined by PCR and immune electron microscopy (IEM): application for HEV detection by affinity-capture RT-PCR

Previously, we have described that injection of an expression vector contai ning hepatitis E virus (HEV) open reading frame 2 (HEV-ORF-2) generated a s trong antibody response in mice. To characterize the reaction of this antis erum with native HEV and to evaluate its potential diagnostic application, we tested the antiserum's ability to bind HEV using immune electron microsc ope (IEM) and affinity-capture reverse transcription polymerase chain react ion (RT-PCR) amplification. Antiserum to ORF-2 aggregated HEV virions as se en by electron microscopy, providing direct evidence that ORF-2 encodes a s tructural protein. Antiserum also captured HEV for RT-PCR amplification. Th is antiserum bound HEV from diverse origins (Asia, Africa, Mexico) at virus concentrations found in patient fecal specimens and bile from inoculated n on-human primates. The specificity of the affinity binding was demonstrated when pre-immune sera or sera collected from mice injected with control DNA vector (lacking the HEV ORF-2 gene) failed to bind HEV for RT-PCR amplific ation and IEM. Specific RT-PCR amplification was confirmed by restriction e nzyme digestion of PCR products. The sensitivity of the binding was evaluat ed by RT-PCR amplification of serially diluted bile containing a geneticall y divergent HEV, Mexico'86. HEV was detected in a 10(-8) dilution of this b ile. This is the first report that antibodies elicited by a DNA vaccine rec ognize native HEV. Our results indicate that ORF-2 encodes a structural pro tein and that antiserum to this protein enables simple, sensitive, and spec ific HEV detection by affinity-capture RT-PCR. (C) 1999 Elsevier Science B. V. All rights reserved.