Jun 4, 1993 - K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for ...
JOURNAL OF VIROLOGY, Nov. 1993, P. 6753-6761 0022-538X/93/1 16753-09$02.00/0 Copyright © 1993, American Society for Microbiology
Vol. 67, No. 11
Characterization of Hepatitis C Virus Envelope Glycoprotein Complexes Expressed by Recombinant Vaccinia Viruses ROBERT RALSTON, KENT THUDIUM, KIM BERGER, CAROL KUO, BARBARA GERVASE, JOHN HALL, MARK SELBY, GEORGE KUO, MICHAEL HOUGHTON,* AND QUI-LIM CHOO Chiron Corporation, 4560 Horton Street, Emneryville, California 94608
Received 4 June 1993/Accepted 13 August 1993 We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (w/HCVI 906) encoding C-El-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (El), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the El and E2 expressed by w/HCV1 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these El and E2 species represent intracellular forms of the HCV envelope proteins. HCV El and E2 formed El-E2 complexes which were precipitated by either anti-El or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between El and E2 was detected. El and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified El-E2 generated high titers of anti-El and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified El-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, 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. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes. 906
Hepatitis C virus (HCV) now is recognized as a major agent of chronic hepatitis and liver disease throughout the world (5, 7, 9, 18, 22). Recognition of the prevalence and importance of HCV as a human pathogen was made possible by the molecular cloning of the virus genome and development of immunoscreening assays with recombinant HCV antigens (7, 22). Molecular cloning of HCV (isolate HCV-1) revealed that it is a positive-strand RNA virus whose genome encodes a single large polyprotein product of 3,011 amino acids (8). The nucleotide and deduced amino acid sequences and hydropathy profile of HCV show local regions of significant similarity to those of flaviviruses and pestiviruses, suggesting that HCV has a similar genome structure (8, 14, 26, 37). These predictions have been borne out by expression of the HCV polyprotein by using the vaccinia virus/T7 expression system (13, 34). Flaviviruses and pestiviruses are important pathogens which cause significant morbidity and mortality in both human and animal populations. However, HCV differs from its relatives and indeed from most other RNA viruses in the high efficiency with which it establishes chronic infections. Approximately 60% of infected individuals develop chronic infections and associated liver disease, including serious sequelae of cirrhosis and hepatocellular carcinoma (9, 32). The reason for the failure of host immune responses to clear HCV infection is not known. Studies of HCV reinfection in convalescent chimpanzees have suggested that natural infection produces very little protective immunity (1 1). For flaviviruses and pestiviruses, the development of neutralizing antibodies is correlated with protective immunity (16, 20, 31), and poor development of neutralizing antibodies to HCV has been proposed as the basis *
for the susceptibility of convalescent chimpanzees to reinfection (10). Because the only assay currently available for infectious HCV requires experimental inoculation of chimpanzees, the role of neutralizing antibodies in control of infection is poorly understood. The relationship between antibodies which recognize recombinant HCV structural proteins and the ability of these antibodies to react with virus also is not known because HCV virions have not been purified and their molecular composition has not been determined. Expression of the structural region of HCV in recombinant systems by several laboratories has demonstrated that the amino-terminal portion of the HCV polyprotein can be processed into a basic protein of 18 to 22 kDa followed by two membrane-associated glycoproteins of 31 to 35 kDa and 70 to 72 kDa which are believed to represent the core (C) and envelope (El and E2) proteins of the virus (13, 17, 29, 34, 36). However, the HCV structural proteins have not been characterized extensively and studies of the intramolecular association between El and E2 have produced conflicting results (13, 27). In order to understand the immunology and pathogenesis of HCV and to develop a vaccine, we will need to generate expression systems and purification methods which produce HCV proteins with native antigenic structure (28). In the case of flaviviruses and pestiviruses, recombinant vaccinia virus vectors have proven to be useful for expression of viral proteins which clicit neutralizing antibodies (2, 10, 20, 25, 30, 31, 44). We previously have described the use of recombinant vaccinia viruses containing all or part of the HCV structural region for analysis of anti-HCV cytotoxic T-cell responses (21). We describe here the characterization and purification of HCV envelope glycoproteins expressed by such recombinant vaccinia viruses and demonstrate the immunogenicity of these
Corresponding author. 6753
6754
J. VIROL.
RALSTON ET AL. TABLE 1. HCV structural region constructs in recombinant vaccinia virus vectors
Recombinant vacciniia virus
vv/HCV -,0, vv/C-El 1-4
vv59/C-E1
381
vv/E2-NS23470,, vv59/HCV1-90,, vv/tpa-NSI vv/lac
HCV- I codon
HCV- I site
Met-t-Lcu-906
StulI-BglIL"
Met-l-Ile-340 Met-l-Val-381 Met-347-Leu-906 Met-l-Leu-906 Gly-406-Glu-661
Stuil-BamiiHI StulI-Sall (blunt) BamHI-BglIIl
AptLI-XbaI PCR
None
Vector
Vector
codoni
Vector sitc
pSC 11 pSCI 1 pSC59 pSCI 1 pSC59 pSCl I pSCII
None Nonc LLVK None None tpa lcader'
Sinal-3glII
SSmaI-BglII StllI BglII Stiul-SpeI Asp7l8-Bgl1l
This constrLiCt contains a synthetic in-frame stop codon and f3,lll site immediately 3' to codon 906 (TTG). /'A sequLence encoding the tpai signil peptide (36) was linked in framc 5' to HCV codon 40)6 (GGC).
"
proteins in chimpanzees. The ability of these recombinant envelope proteins to generate protective immunity to HCV will be described in detail separately. MATERIALS AND METHODS Construction of recombinant vaccinia viruses that express HCV structural proteins. A complete cDNA copy of the HCV structural region was reconstructed from fragments of the prototype HCV-l cDNA cloned in phage Xgtl 1 (7, 8). The accuracy of the reconstructed cDNA sequence was verified by DNA sequencing. Recombinant vaccinia viruses were generated with the pSCl 1 promoter-transfer vector (3), which had been modified by insertion of a polylinker at the SmaI site (SmaI-KpnI-Bg1II-HindIII; gift from R. Sekulovitch). For purification of HCV-1 envelope glycoproteins, recombinant vaccinia viruses werc also prepared with the promoter-transfer vector pSC59 (gift from S. Chakrabarti and B. Moss), which contains a synthetic hybrid early-late promoter designed to increase expression of recombinant proteins compared with that obtained with the P7.5 promoter in pSCI 1. The constructs used for expression of HCV-1 structural proteins are summarized in Table 1. Cells and viruses. Vaccinia virus strain WR (ATCC VR119) and cell lines BSC-40, 143B (ATCC CRL 8303), and HeLa S3 (ATCC CCL 2.2) were obtained from B. Moss (National Institutes of Health). Cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g of D-glucose (J. R. Scientific) per liter supplemented with 10% fetal bovine serum (HyClone), 50 p.g of penicillin per ml, and 5 [ig of streptomycin per ml (J. R. Scientific). Recombinant vaccinia viruses incorporating HCV genes were prepared according to the method of Chakrabarti et al. (3). Antisera and immunoprecipitation. Immune sera from an HCV-seropositive human subject (42) were used for immunoprecipitation of HCV envelope proteins as described previously (36). Affinity-purified rabbit hyperimmune sera raised against core, El, and E2 antigens expressed in Saccharomyces cerevisiae have been described previously (34). For presentation of data, all composite photographs represent data obtained from single gels. For immunoprecipitation, 106 BSC-40 cells were infected with recombinant vaccinia viruses at a multiplicity of infection of 2 for 16 h and then starved for methionine and cysteine for 1 h in Dulbecco's modified Eagle's medium minus methionine and cysteine. The medium then was changed and the cells were labeled for 4 h in Dulbecco's modified Eagle's medium minus methionine and cysteine, supplemented with 100 pCi each of [35S]methionine and [355S]cysteine (>1,200 and > 1,700 Ci/ mmol, respectively; New England Nuclear). After labeling, cells were washed once with Dulbecco's phosphate-buffered
saline (PBS) solution (J. R. Scientific) and then lysed in 1 ml of lysis buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.5], 1 mM Na EDTA, 0.5C Triton X-l00). Nuclei and debris were pelleted at 14,000 x g at 4°C for 5 min in an Eppcndorf microcentrifuge, and HCV-specific proteins were immunoprecipitated from the clarified lysates by using protein-A-Sepharose-bound human or rabbit antibodies which previously had been blocked with clarified lysates prepared from equivalent numbers of BSC-40 cells infected with a control vaccinia virus expressing P-galactosidase. Protein A-Sepharose-bound immune complexes were washed four times with lysis buffer and then twice with 120 mM Tris-Cl (pH 7.0) prior to resuspension in I x sample buffer (23) and electrophoresed through sodium dodecyl sulfate (SDS)-10% polyacrylamide gels. Western blotting (immunoblotting). Analysis of proteins bound to polyvinylidene difluoride membranes (Immobilon-P; Milliporc) was performed according to the method of Towbin et al. (40) after transfer of proteins in Tris-glycine-SDS buffer at 10 V for 15 h. Blots were processed as described previously (40), using either HCV+ patient serum diluted 1:200, rabbit anti-core-protein (C22 antigen), anti-El (S2 antigen), or anti-E2 (NS 1-antigen) antibody diluted 1:500 (34) or chimpanzee anti-E1-E2 serum diluted 1:400 as the primary antibody and either goat anti-human immunoglobulin or anti-rabbit immunoglobulin conjugated to horseradish peroxidase (BoehringerMannheim) diluted 1:25,000 as the secondary antibody. Reactive proteins were detected by enhanced chemiluminescence (ECL; Amersham) according to the manufacturer's protocol. Subcellular localization of envelope glycoproteins. BSC-40 monolayers were infected with w/HCV1t,,I, and labeled as described above. Labeled cells were washed once with PBS, scraped into PBS, and pelleted in a variable-speed microcentrifuge at 82 x g for 5 min at 4°C. Cells were resuspended in a hypotonic buffer (20 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid [HEPES] [pH 7.4], 10 mM NaCl, 1 mM MgCl,) and disrupted by passage through a 27-gauge hypodermic needle 25 to 30 times. Nuclei were collected by centrifugation at 1,000 x g for 5 min at 4°C. The supernatant was recentrifuged at 20,000 x g for 30 min at 4°C to yield a microsomal fraction (P-20) and a cytosolic fraction (S-20). All fractions were adjusted to 1 x in lysis buffer, passed several times through a 27-gauge needle, and clarified before immunoprecipitation with HCV+ patient serum. To further characterize the solubility of the envelope glycoproteins which were found to be retained in the P-20 fraction, fresh P-20 pellets were prepared and further extracted in either a high-salt buffer (0.5 M NaCl, 10 mM EDTA) or a borate buffer (20 mM sodium borate [pH 9.0], 10 mM EDTA) by trituration through a 27-gauge needle. Membrane extracts were recentrifuged as described above, and the resulting pellets and supernatants
Vol. 67, 1993
were adjusted to 1 x in lysis buffer and immunoprecipitated as described above for the initial P-20 and S-20 fractions. Lectins and glycosidases. For precipitation with glycanspecific lectins, proteins were radiolabeled as described above and extracted in lysis buffer supplemented with 1 mM (each) CaCl, MgCl,, and MnCI,. Radiolabeled proteins then were precipitated with biotinylated lectins (Boehringer-Mannheim) bound to avidin-acrylic beads (Sigma). The biotinylated lectins (agglutinins) used for these studies were from snowdrop (Galantthius niivaluts; GNA), elderberry (Sanmbulclis niger; SNA), peanut (PNA), wheat germ (WGA), and jack bean (ConA). Purification of El and E2. One-liter cultures of HeLa S3 cells were grown to a density of 5 x 1(P cells per ml in spinner cultures of Dulbecco's modified Eagle's medium supplemented with 5% calf serum and 100 jig each of penicillin and streptomycin sulfate. Cells were infected with recombinant vaccinia virus w59/HCV)9 ()O at a multiplicity of infection of 2 and grown for 15 h. All further steps were carried out at 0 to
40C. Infected cells were collected by centrifugation at 1,000 x g for 10 min, washed once with PBS, and lysed with 50 ml of lysis buffer (100 mM NaCl, 20 mM Tris-HCI [pH 7.5], 1 mM EDTA, 0.5cc Triton X-100). The lysate was clarified by centrifugation at 20,000 x g for 15 min and applied to a GNA-agarose column (1 by 5 cm; Vector Laboratories). The column was washed extensively with lysis buffer, and the bound proteins were eluted in 0.5-ml fractions by using 0.9 M cx-D-mannopyranoside in lysis buffer. Column fractions containing HCV-l envelope proteins were pooled and diluted 10-fold in lysis buffer minus NaCI and then applied to a DEAE-Fractogel column (0.5 by 10 cm). The flowthrough fraction containing the HCV envelope glycoproteins was concentrated by ultrafiltration on Amicon YM-30 membranes and stored at -70°C. For immunization studies, E1-E2 preparations were UV irradiated at 200 nm for 12 s (36 pJ/cm2) in a UV cabinet (Stratagene) to ensure the absence of live recombinant vaccinia viruses. The titers of vaccinia virus in E1-E2 preparations were
