1 Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Ave, Athens 115 21, Greece
2 The Edward Jenner Institute for Vaccine Research, Compton, UK
3 Xenova Group plc, Berkshire, UK
4 Second Department of Medicine, Athens University School of Medicine, Greece
5 Nuffield Department of Medicine, University of Oxford, Oxford, UK
Correspondence
Penelope Mavromara
penelopm{at}hol.gr
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ABSTRACT |
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Present address: Nuffield Department of Medicine, University of Oxford, Oxford, UK.
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INTRODUCTION |
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The HCV genome encodes two putative envelope glycoproteins, E1 and E2 (Reed & Rice, 2000). Both glycoproteins are thought to be type I transmembrane proteins that accumulate in the endoplasmic reticulum (ER) and interact non-covalently to form a heterodimer (Duvet et al., 1998
). Glycoprotein folding and formation of non-covalently associated E1E2 complexes are slow and inefficient processes that occur in association with the ER-resident chaperone calnexin (Choukhi et al., 1998
). The folding of E2 is independent of E1 but E2 is required for the proper folding of E1, suggesting that E2 acts as a chaperone for the folding of E1 (Cocquerel et al., 1998
). The E2 protein is (depending on the genotype) a 363370 amino acid (aa) glycoprotein (5874 kDa) with 20 conserved cysteines and 9 potential N-glycosylation sites (Depraetere & Leroux-Roels, 1999
). At its carboxyl terminus, E2 has a hydrophobic anchor (aa 718746) that retains the native E2 glycoprotein in the ER (Duvet et al., 1998
). Truncated E2-proteins lacking this region are soluble and secreted upon expression in mammalian cells (Michalak et al., 1997
; Mizushima et al., 1994
).
Owing to the lack of an in vitro system to propagate the virus and thus of sufficient quantities of HCV virions, truncated soluble mimics of E2 have been analysed for their potential use in the study of virushost interactions and/or vaccine development (Depraetere & Leroux-Roels, 1999). In particular, a secreted form of E2 from HCV strain H truncated at aa 661 (E2-661) has been shown previously to react with a panel of conformation-sensitive antibodies, indicating that E2-661 is folded in a conformation comparable to the E2 in the native E1E2 complex (Deleersnyder et al., 1997
; Flint et al., 2000
). Most importantly, E2-661 was found to bind to CD81, a host cell-surface protein suggested to play a role in the virus attachment/entry process (Pileri et al., 1998
). Thus, truncated forms of the E2 protein may be useful reagents for in vitro immunobiological studies, vaccine development and generation of HCV diagnostics. Furthermore, recombinant chimeric E2 proteins have been generated through the replacement of the ER anchor with the transmembrane domain of other proteins, such as from vesicular stomatitis virus (Buonocore et al., 2002
), influenza A virus haemagglutinin (Flint et al., 1999
), CD4 (Cocquerel et al., 1998
; Siler et al., 2002
) or platelet-derived growth factor receptor (Forns et al., 1999
) in order to express the truncated from of E2 on the cell surface.
To optimally produce high quantities of correctly folded and glycosylated antigens, a large number of systems have been exploited for the expression of HCV glycoproteins (Depraetere & Leroux-Roels, 1999; Op De Beeck et al., 2001
). Prokaryotic and yeast expression systems lead to high-level expression of non-glycosylated proteins (Lok et al., 1993
; Mita et al., 1992
; Yokosuka et al., 1993
), and approaches using baculovirus-infected insect cells produce large quantities of partially glycosylated antigens (Lanford et al., 1993
; Matsuura et al., 1992
). Expression in eukaryotic systems has been based on transient or stable transfections of cells with DNA vectors (
Chien et al., 1993; Zaaijer et al., 1994
) or infections with recombinant viruses (Buonocore et al., 2002
; Siler, 2002
; Dubuisson et al., 1994
; Fournillier-Jacob et al., 1996
). Eukaryotic expression systems are believed to yield native, properly glycosylated antigens, but only recombinant viruses combine high quantity with satisfactory quality. Another limitation of the eukaryotic expression systems is their tendency to generate disulfide-linked E2 aggregates, which are most likely non-functional dead-end products, yet may play a role in viral escape mechanisms (Choukhi et al., 1999
; Dubuisson, 1998
). This occurs primarily after DNA transfection (Flint et al., 2000
) but also, to a variable degree, in certain viral expression systems (Michalak et al., 1997
).
In this study we evaluate, for the first time, the potential use of a recombinant herpes simplex virus type 1 (HSV-1)-based vector for expression of a truncated form of the HCV E2 from genotype 1a. HSV-1 is a large double-stranded neurotropic DNA virus and is a common human pathogen (Roizman & Knipe, 2001; Whitley & Roizman, 2001
). Nevertheless, recent extensive studies have shown that the new generation of defective HSV-1 recombinants (both replication-defective or replication-competent attenuated viruses) are suitable expression vectors with potential applications in gene therapy or vaccine development (Brockman & Knipe, 2002
; Da Costa et al., 2001
; Latchman, 2001
). Here, we used a replication-defective HSV-1 recombinant which lacks the UL22 gene encoding the essential glycoprotein H (gH) (Forrester et al., 1992
). Complementing CR1 cells expressing gH are required for the propagation of the recombinant virus as gH is required for HSV entry into the target cells. Viruses produced from the complementing cell line can infect normal cells, but the virus is restricted to a single round of replication since the resulting virus progeny lacks gH and is therefore non-infectious. The gH-deleted HSV-1 recombinant, also known as a disabled infectious single cycle (DISC) herpes simplex virus, is a well-characterized viral vector system with extensive pre-clinical safety data (Loudon et al., 2001
). In addition, by virtue of its capacity for a single round of replication in infected cells, the DISC-HSV virus is predicted to support efficient expression of a foreign gene.
In this study we present evidence indicating that the DISC-HSV recombinant virus efficiently expresses non-aggregated, immunobiologically active intracellular and secreted forms of the E2-661 protein and induce a specific Th1-type antibody response against HCV E2 protein in a murine system.
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METHODs |
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Antibodies.
MAbs H33, H47 and H53 were kindly supplied by Jean Dubuisson (Institute Pasteur, Lille, France) (Cocquerel et al., 1998; Deleersnyder et al., 1997
). Hybridoma cell lines producing rat anti-E2 mAbs 3/11, 6/53 and 1/39 were kindly provided by Jane McKeating (University of Reading, Reading, UK). Hybridoma supernatants were used without further purification.
Generation and propagation of DISC-HCV recombinant viruses.
DISC-HSV-1 strain 17 was kindly supplied by Xenova. Plasmid pIMJ28 (kindly supplied by Xenova) is a shuttle vector designed to contain two expression cassettes. The first expression cassette contains an open reading frame (ORF) encoding the green fluorescent protein (GFP) under the control of the Rous sarcoma virus promoter. GFP serves as a marker gene and is used for the selection and purification of recombinant viruses. The second cassette consists of an immediate early cytomegalovirus (ieCMV) promoter followed by a multiple cloning site in which the gene of interest is inserted, a poly(A) tail and stop codons in all reading frames.
E2 sequences were amplified from a vector containing the full-length cDNA sequence of the HCV H strain (genotype 1a), kindly provided by Genevieve Inchauspe (INSERM, Lyon, France).
For DISC-gEE2661, an ORF consisting of the HSV gE leader sequence (HSV nt 141183141309) serving as a signal sequence and aa 384661 of HCV was subcloned in-frame into pIMJ28. All constructs were confirmed by sequencing and a sequence change at aa 370 leading to a substitution of glutamic acid for lysine was corrected. A DISC-GFP, which encodes the GFP under the control of the ieCMV promoter, was constructed as a mock-infection control.
DISC-HCV recombinants were constructed using the PacI-based ligation technique as previously described by Xenova (Loudon et al., 2001). The ligation mix was transfected into gH-expressing CR1 cells using Lipofectamine (Life Technologies). Recombinant GFP fluorescent viruses were purified using limiting dilution assays and single plaques were screened by PCR using E2-specific primers and verified by Southern blots (Sambrook, 1989
). DISC-HCV recombinants were maintained and titrated in CR1 cells.
Standard infection, metabolic labelling and immunoprecipitation.
Confluent monolayers of Vero or CR1 cells were grown in six-well plates (Corning Costar) or 25 cm2 flasks and were inoculated with 5 p.f.u. in 199V medium containing 1 % FBS (Life Technologies). After 2 h adsorption at room temperature, the medium was replaced with fresh 199V medium containing 1 % FBS and infection allowed to proceed.
For enzyme immunoassay (EIA), supernatant was collected after 20 h, clarified by centrifugation and concentrated 60-fold using Centripreps (10 000 kDa cut-off; Amicon). The infected cells were pelleted by centrifugation and resuspended in 3 ml 0·15 M NaCl/0·5 M Trizma Base (Sigma) and complete proteinase inhibitor (Boehringer Mannheim) at pH 8·8. Cells were disrupted by sonication for 3 min. Cellular debris was removed by centrifugation.
For labelling with [35S]methionine, Vero or HepG2 cells were washed 6 h post-infection (p.i.) with pre-warmed DMEM without methionine (Life Technologies) and then incubated in the same medium for 1 h to induce methionine starvation. For steady-state labelling, infected cells were labelled for the indicated time with 3050 µCi [35S]methionine (ICN Biochemicals) ml-1, in medium containing 1/40 of the normal concentration of methionine. After labelling, supernatant was collected and infected cells were washed once with ice-cold PBS before lysis (20 min/4 °C) in Tris-buffered saline (TBS) lysis buffer containing 0·5 % Igepal (Sigma). Cell lysates and supernatants were clarified by centrifugation (15 min/4 °C/12 000 g) and then precleared with Protein ASepharose after TBS and Igepal concentrations had been adjusted to 1x times; and 0·2 % respectively. Immunoprecipitations were carried out as previously described (Dubuisson et al., 1994). The precipitates were solubilized by heating for 3 min at 96 °C in SDS-PAGE sample buffer and run on a 10 or 12·5 % polyacrylamide gel. Separated proteins were transferred onto a nitrocellulose membrane (Protran BA nitrocellulose, Schleicher and Schuell) and exposed to Kodak XAR film.
Endoglycosidase digestion.
Immunoprecipitated proteins were eluted from Protein ASepharose beads in 0·5 % SDS and 1 % mercaptoethanol by heating at 96 °C for 10 min. The protein eluates were then divided into three equal portions for digestion with peptide N-glycosidase (PNGase) F or endo--N-acetylglucosaminidase H (Endo H; both enzymes from New England Biolabs) plus an undigested control. Digestions were carried out for 1 h at 37 °C following the manufacturer's protocol. Digested samples were analysed by SDS-PAGE and autoradiography as described above.
Production of secreted E2-661 by transient transfection of cells with an E2 expression plasmid.
Production of secreted recombinant E2-661 by transient transfection of HEK 293 cells was done as described previously (Flint et al., 1999). Briefly, HEK 293 cells were cultured in 145 mm dishes and transfected with a eukaryotic expression vector encoding E2-661 (p14.tE2·661.hiv, HCV genotype 1a, kindly provided by J. McKeating) using Effectene (Qiagen). Supernatant was harvested after 72 h. E2 was enriched from these supernatants using GNA lectin columns (Amersham Pharmacia) or affinity columns, produced by coupling purified anti-E2 antibodies (1/39 or H33) to HiTrap NHS activated columns (Amersham Pharmacia).
Western blot analysis.
Samples were separated by SDS-PAGE on 10 % or 12·5 % polyacrylamide gels under reducing or non-reducing conditions. Proteins were then transferred to nitrocellulose membranes (Protran BA Nitrocellulose) and probed with either anti-E2 mAb H47 (a mouse mAb, detected using a peroxidase-conjugated goat anti-mouse IgG; Pierce) or anti-E2 mAb3/11 (a rat mAb, detected using a peroxidase-conjugated anti-rat IgG; Harlan Sera-Lab). Proteins were then visualized using an enhanced chemoluminescence system (Amersham Pharmacia).
The amount of intracellular versus secreted antigen was estimated by EIA, using several dilutions. All samples were analysed on the same plate. Using this analysis, the amount of intracellular antigen corresponded to the amount of secreted antigen of the same preparation when cell pellets were resuspended in an equivalent volume as the concentrated supernatant.
GNA-capture EIA and CD81-capture EIA.
EIAs were performed as previously described (Flint et al., 2000). Captured antigen was detected by incubation with the conformation-dependent anti-E2 mAb H53 followed by incubation with an anti-mouse IgG1HRP antibody (BD/Pharmingen) and a tetramethylbenzidine substrate (TMB; Sigma). As a positive control for CD81 binding to the plates, an anti-CD81 mAb (Autogen-Bioclear) that recognizes both human and mouse CD81 was used in conjunction with an anti-goat IgGHRP mAb (Santa-Cruz Biotechnology). Absorbance values were determined at 450 nm using an EIA reader (SpectraMax 340, Molecular Devices, UK).
Sera.
Coded sera from 55 individuals were tested for reactivity against secreted E2-661. They were obtained from: 38 patients, 19 males and 19 females, 1870 years old with chronic hepatitis C (CHC); 7 patients with other forms of liver disease, i.e. primary biliary cirrhosis (PBC), chronic hepatitis B (HBV) and chronic hepatitis G (HGV); and 10 healthy individuals negative for anti-HCV antibodies. All sera from patients with CHC were obtained before the beginning of interferon- treatment and were anti-HCV positive by EIA (Ortho) and positive for HCV RNA (Roche, Amplicor). The infected genotype was determined by INNOLiPA (Innogenetics). According to their response to interferon therapy they were divided into three groups: (a) sustained responders (SR) with negative HCV RNA in serum and normal alanine aminotransferase (ALT) levels at the end of the treatment, as well as at the end of a 6 month follow-up after cessation of therapy, (b) relapsed responders (RR) with negative serum HCV RNA and normal ALT levels at the end of therapy, but with virological and biochemical relapse during the 6 months of post-treatment follow-up; and (c) non-responders (NR) with positive serum HCV RNA and increased ALT levels maintained up to the end of interferon-
therapy.
Detection of HCV antibodies in serum samples.
Immunolon II EIA plates (Dynal) were coated with 50 µl per well of the secreted or intracellular recombinant E2-661 antigen, respectively, in TBS at an estimated final concentration of 0·5 µg per well and incubated for 1 h at 37 °C. Wells were blocked with 100 µl TBS supplemented with 5 % w/v non-fat milk for 1 h at room temperature and subsequently washed once with TBS containing 1 % (w/v) non-fat milk and 0·1 % (v/v) Tween 20 (TBS-MT). Patient sera diluted 1/100 in TBS-MT were preincubated with HSV glycoproteins (5 µg ml-1) for 1 h at 37 °C in order to remove anti-HSV-specific antibodies from the samples. Pretreated serum samples (100 µl) were added to each well and the plates were incubated for 1 h at 37 °C. After washing, 100 µl rabbit anti-human IgG conjugated to HRP (Dako) at 1/1000 in TBS-MT solution was added to each well and the plates were incubated for 1 h at 37 °C. The final immune complexes were detected by addition of 100 µl TMB substrate solution and colorimetric determination of the absorbance at 450 nm. For each sample, the final absorbance value was calculated by subtracting the A450 of the control antigen EIA from the respective A450 of the fusion protein EIA. The cut-off point was determined as the mean value of the negative samples plus two standard deviations (SD).
Immunization procedures.
The housing, maintenance and care of the animals were in compliance with all relevant guidelines and requirements. Groups of three to five female BALB/c mice (68 weeks old) were injected twice at day 0 and 14 intraperitoneally with 5x106 p.f.u. of DISC-gEE2661 recombinant virus, whereas control animals received 5x106 p.f.u. DISC-GFP virus.
Antibody analysis.
Sera collected from the immunized animals at the indicated times were analysed for anti-E2 total IgG Ab concentration by standard EIA methodology, using prokaryotic E2 recombinant protein isolated from inclusion bodies. Medium binding EIA plates (Corning Costar) were therefore coated with prokaryotic E2-antigen at a concentration of 0·5 µg per well. Total IgG was detected in mice sera by HRP-conjugated anti-mouse immunoglobulins (Dako) as a secondary antibody and developed with TMB substrate. Analysis of the antibody isotypes was performed according to the manufacturer's instructions using a commercial isotyping kit (BD/Pharmingen).
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RESULTS |
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To analyse the glycosylation status of the HCV E2-661 protein expressed in the context of the HSV-1 viral vector, Vero cells were infected with the DISC-gEE2661 virus for 10 h and were subjected to [35S]methionine labelling for the last 4 h of infection. Cell lysates and supernatants were collected and digestions with Endo H and PNGase F on the immunoprecipitates were carried out as described. Endo H removes the chitobiose core of high mannose and some hybrid forms of N-linked sugars but not complex forms of N-linked sugars, whereas PNGase F removes high-mannose and complex sugars. Therefore, resistance to digestion with Endo H but not with PNGase F indicates that the proteins reached the trans-Golgi where complex sugars are added. The analysis of the glycosylation pattern is shown in Fig. 2. Only the secreted E2-661 protein expressed in Vero (Fig. 2b
, lanes 46) or HepG2 cells (Fig. 2c
, lanes 46) was resistant to Endo H digestion. In contrast, the intracellular form of E2-661 was Endo H sensitive (Fig. 2b, c
, lanes 13). These results confirm that the secreted E2-661 protein undergoes Golgi processing prior to secretion when expressed in the context of HSV infected cells.
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Antibody response in mice vaccinated with the recombinant DISC-gEE2661 virus
To analyse the immunogenicity of the DISC-HCV recombinant virus, BALB/c mice were injected intraperitoneally with 5x106 p.f.u. of DISC-gEE2661 or the parental DISC-GFP virus twice (on days 0 and 14) and the E2-specific IgG responses were measured by reactivity against plate bound recombinant E2 by EIA at days 14 and 28 post-initial (day 0) injection. As shown in Fig. 6(a), all mice immunized with recombinant DISC-gEE2661 virus developed high levels of anti-E2 antibodies after the first immunization which increased further following the second inoculation. No antibodies were detected in mice immunized with the control virus. Subsequent subclass analysis of the anti-E2 antibodies showed that the predominant anti-E2 isotype was IgG2a, suggesting a strong Th1-like response. The IgG1 levels were significantly lower (Fig. 6b
). Notably, intramuscular immunization with a DNA vaccine 14 days prior to inoculation with the DISC-gEE2661 virus improved the levels of the IgG1 isotype (data not shown).
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DISCUSSION |
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As mAb H53 reactivity has been correlated with an E2-conformation capable of binding to CD81 (Flint et al., 2000), we also investigated whether intracellular and secreted E2-661 produced by DISC-HCV recombinants was capable of binding to CD81 by using an EIA-based capture assay. Consistent with results previously obtained using E2-661 produced using a transient transfection system (Flint et al., 2000
), we found that both intracellular and secreted forms of E2-661 were able to bind to recombinant CD81 in a concentration-dependent manner, and that intracellular E2 bound nearly twice as efficiently to CD81 as the secreted form. The greater efficiency of binding of the intracellular form of E2-661 to CD81 is thought to be due to the differences in glycosylation between this and the secreted form of E2. Secreted E2-661 passes through the Golgi compartment following the secretory pathway, a process that leads to the acquisition of complex sugars which might then interfere with CD81 binding either directly or by steric hindrance.
Consideration of how the conformation and glycosylation state of recombinant E2 proteins may be related to forms of the protein that are expressed during in vivo infection with HCV is of importance with regard to the choice of antigen used both for diagnostic tests and in subunit vaccine design. For diagnostic tests, it is important that the E2 protein used should be recognized by sera from infected individuals. Here, we report that secreted E2-661 antigen was recognized in an EIA assay by anti-HCV antibodies present in the sera of patients persistently infected with HCV (79 %). This is comparable to previous data which have shown that most chronic HCV-sera (8097 %) contain IgG antibodies reactive with recombinant E2 antigen produced using mammalian expression systems (Chien et al., 1993; Mink et al., 1994
; Saracco et al., 1994
; Yuki et al., 1996
). Anti-E2 specific antibodies are much less efficient at recognition of E2 antigen produced in insect cells (Chien et al., 1992
; Hussy et al., 1997
; Lanford et al., 1993
), yeast (Chien et al., 1993
; Lok et al., 1993
; Mink et al., 1994
) or bacteria (Hussy et al., 1997
; Mita et al., 1992
; Yokosuka et al., 1993
), indicating an important role for protein conformation and glycosylation in antibody recognition. As previously described, an individual's response to interferon therapy was not predictable on the basis of the anti-E2 antibody level measured before treatment (Fournillier-Jacob et al., 1996
; Saracco et al., 1994
). No false positive results in the controls of uninfected, HBV- or HGV-infected patients, nor in sera of patients with autoimmune cirrhosis (primary biliary cirrhosis), were observed. Furthermore, we compared the ability of human sera from a limited number of infected individuals to recognize the intracellular versus secreted forms of E2-661. Interestingly, some of the sera could discriminate between the two forms of the protein, suggesting that a proportion of infected individuals have an antibody response to epitopes that are differentially expressed in the two different forms of the protein. A previous study also documented preferential reactivity of serum samples from a proportion of infected individuals with the intracellular from of E2 (Flint et al., 2000
), and suggests that an EIA assay combining intracellular and secreted forms of E2 may be beneficial for diagnostic purposes.
Although the nature of the protective immune responses against HCV is not well defined, several studies suggest that HCV E2 protein represents a potential target for vaccine development (Abrignani & Rosa, 1998; Choo et al., 1994
; Siler et al., 2002
; Zucchelli et al., 2000
). To this end, we present preliminary evidence that vaccination of BALB/c mice with a replication-defective HSV recombinant virus expressing a truncated HCV E2 protein (DISC-gEE2661) is capable of eliciting a strong humoral immune response against the E2 protein. It is noteworthy that all animals developed high levels of E2-specific antibodies after the first immunization, which increased further following the second immunization. Isotype analysis of the response against the E2 antigen suggested a strong Th1-type response by the almost exclusive detection of IgG2a. Therefore, the use of attenuated recombinant HSV-based vectors provides an alternative virus-based expression system efficient at producing large quantities of non-aggregated, correctly folded, seroreactive, CD81-binding recombinant protein and may represent a new immunization strategy to induce humoral and cellular immune responses against HCV. Experiments characterizing the cellular immune response are in progress to further evaluate such possibilities.
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ACKNOWLEDGEMENTS |
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Received 14 August 2002;
accepted 6 November 2002.