CHARACTERIZATION OF HEPATITIS-C VIRUS ENVELOPE GLYCOPROTEIN COMPLEXESEXPRESSED BY RECOMBINANT VACCINIA VIRUSES

We constructed recombinant vaccinia virus vectors for expression of th e structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated majo r protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa ( E2), as observed previously with other mammalian expression systems. T he bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, su ggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedime nted at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agar ose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E 2 generated high titers of anti-E1 and anti-E2 antibodies. Further stu dies, to be reported separately, demonstrated that purified E1-E2 comp lexes were recognized at high frequency by HCV+ human sera (D. Y. Chie n, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo , Lancet, in press) and generated protective immunity in chimpanzees ( Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. H an, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. C hing, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes.