CHARACTERIZATION OF HCV AND ANTI-HCV

尽管缺乏对丙型肝炎病毒再感染的保护性免疫

Although the lack of protective immunity against reinfection with HCV has been reported, our safety study in a chimpanzee infused with an HCV RNA positive intravenous immune globulin (IGIV) prepared from a c100-3 reactive plasma pool and other recent studies support the existence of neutralizing antibodies toward envelope proteins for HCV. To assess the presence of anti-E1 and anti-E2 in certain plasma and IGIV preparations, we partially purified recombinant E1 and E2 proteins produced in a baculovirus eukaryotic expression system. Cells infected with baculovirus were subjected to freeze-thawing, sonication, and solubilization with 8 M urea. After dialysis, the solution was applied to a GNA (a lectin)-agarose column to bind glycosylated proteins. The GNA bound proteins were eluted, analyzed on a gradient SDS-polyacrylamide gel, transferred to a nitrocellulose paper, and immune blotted. A monoclonal anti-E1 identified two prominent bands, 30 kd (E1) and 105 kd, while a monoclonal anti-E2 identified one major band, 70 kd and a faint band, 105 kd. Based on the construct of the recombinant clone, the 70 kd protein is an E2-polyhedrin fusion protein while the 105 kd protein may be an unprocessed E1-E2-polyhedrin, a complex between the two, or a multimer of E1. Thus, GNA bound proteins were used to set up ELISAs for detection of antibodies to E1 and E2 in hybridoma cell cultures, human sera or plasma, or immune globulin preparations. The specificity for our newly developed ELISA utilizing a peroxidase conjugated anti-mouse IgG as a second antibody to detect monoclonal antibodies can be demonstrated since neither a monoclonal anti-core nor a normal mouse serum reacted. A similar ELISA was also set up to detecting human anti-E1 and anti-E2 by using a peroxidase conjugated anti-human IgG. However, in both ELISA and immunoblot, we found that normal human sera had some cross reactivities unless they were further diluted. Although both assays remain to be further improved with respect to its specificity and sensitivity, our preliminary data indicated that the IGIV prepared solely from either anti-c100-3 reactive plasma or multiantigen anti-HCV reactive plasma had higher levels of antibodies to HCV envelop proteins than those IGIV prepared from multiantigen screened plasma.