Amino Acids 1055 to 1192 in the S2 Region of Sever
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病菌学杂志 2005年第6期
Institute of Molecular and Cell Biology, Singapore
Wuhan Institute of Biological Products, Wuhan, People's Republic of China
ABSTRACT
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
INTRODUCTION
A novel coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS) (8, 9, 15, 20). CoVs are positive-strand RNA viruses, and the virion consists of a nucleocapsid (N) core surrounded by an envelope containing three membrane proteins, spike (S), membrane (M), and envelope (E), that are common to all members of the genus (for reviews, see references 16 and 24). The S protein, which forms morphologically characteristic projections on the virion surface, binds to host receptors and mediates membrane fusion. The M and E proteins are important for viral particle assembly, while N is important for viral RNA packaging.
The S protein of CoV is a type 1 integral membrane glycoprotein. It is cotranslationally glycosylated and oligomerized at the endoplasmic reticulum. Its N-linked high-mannose side chains are trimmed and modified and become endoglycosidase H (EndoH) resistant during the transportation to the Golgi apparatus. For some but not all CoVs, the S protein is cleaved into the N-terminal S1 and C-terminal S2 subunits, which contain receptor binding and membrane fusion domains (10, 32), respectively. The mature forms of S are assembled into virions, which release from infected cells. A portion of S is transported to the plasma membrane, resulting in cell-cell fusion or formation of syncytia. The S protein belongs to the class 1 viral fusion proteins and contains two heptad repeat domains (HR1 and HR2) in S2 or the C-terminal region. These two domains interact and play a crucial role in mediating virus-cell membrane fusion and entry of virus into cells.
The S protein of CoV is known to be responsible for inducing host immune responses and virus neutralization by antibodies (5, 7, 14, 25). For SARS-CoV, it has been demonstrated that prior infection provided protective immunity in a mouse model and that the passive transfer of neutralizing antibodies to naive mice also protected them from infection (26). This suggests that neutralizing antibodies have an important role in reducing the viral load or preventing viral replication. A DNA vaccine encoding the S protein alone induces T-cell and neutralizing antibody responses and protected mice from SARS-CoV infection (34), suggesting that the S protein is indeed the primary target for viral neutralization in SARS-CoV infection. This finding was also confirmed by at least four independent studies that use a carrier virus to express S in mice or primates (2, 5, 6, 11). These reports highlight the potential for developing peptide-based vaccine candidates if neutralizing epitopes of S could be identified.
In this study, we aim to identify neutralizing epitopes in the S protein of SARS-CoV, which may be used for the development of vaccine or therapeutic agents against SARS-CoV infection. We expressed different regions of S as glutathione S-transferase (GST) fusion proteins and used them to raise antibodies in rabbits. These polyclonal antibodies were then tested for their specificities for the native and denatured forms of S in Western blot, immunoprecipitation, and immunofluorescence analyses as well as for their capacities to inhibit SARS-CoV replication in Vero E6 culture. A novel neutralizing epitope in S2 containing HR2 was identified.
MATERIALS AND METHODS
Cells and viruses. The monkey kidney cell lines Cos7 and Vero E6, used in this study, were purchased from the American Type Culture Collection (Manassas, Va.). Cos7 and Vero E6 cells were cultured at 37°C in a 5% CO2 incubator in Dulbecco modified Eagle medium containing 0.1 mg of streptomycin/ml, 100 U of penicillin/ml, and 10% fetal bovine serum (HyClone, Logan, Utah). SARS-CoV viral strain Sin2774 (GenBank accession no. AY283798) (22) and recombinant vaccinia virus vTF7-3 expressing bacteriophage T7 RNA polymerase were grown in Vero E6 cells.
Construction of plasmids. (i) Plasmids for expression in mammalian cells. Viral RNA of Singapore strain Sin2774 was extracted and used for amplification of the S gene by reverse transcription-PCR (RT-PCR) with specific primers. The PCR products were digested with BamHI and StuI and ligated into BamHI/EcoRV-cut pKT0, resulting in plasmid pKT-S under the control of a T7 promoter. Specific primers were designed to amplify a strain Sin2774 sequence from nucleotide positions 21476 to 25171, 25066, 24934, 24415, 24157, and 23866. The six RT-PCR products were digested with BamHI and StuI and ligated to BamHI/EcoRV-cut pKT0 under the control of a T7 promoter, giving rise to plasmids pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, pKT-S16, respectively. These constructs encode the C-terminally truncated S proteins, as shown in Fig. 1.
(ii) Plasmids for expression in Escherichia coli. Five PCR fragments encoding the S1, S2, S3, S9, and S10 fragments (Fig. 1) were obtained with the primers listed in Table 1. All primers used were purchased from Research Biolabs or Proligo Pte Ltd. (Singapore). The PCR products were digested by BamHI/XhoI and ligated into the BamHI/XhoI-cut pGEX4T1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden) to obtain plasmids pGEX-S1, pGEX-S2, pGEX-S3, pGEX-S9, and pGEX-S10 for the expression of GST fusion proteins (Fig. 1). The GST tag was located at the N terminus of the fusion protein.
Purification of the recombinant S proteins expressed in E. coli. Plasmids pGEX-S1, pGEX-S2, pGEX-S3, pGEX-S9, and pGEX-S10 were separately transformed into BL21(DE3) cells. A single colony from each plate was grown at 37°C overnight in Luria-Bertani (LB) medium containing ampicillin (100 μg/ml). Five milliliters of the resulting cultures was inoculated into 2 liters of LB medium containing ampicillin (100 μg/ml), which was incubated in a shaker at 37°C until the optical density at 600 nm reached 0.6. Expression of proteins was induced with 1 mM IPTG (isopropyl-?-D-thiogalactopyranoside). Cells were harvested 2 h after induction by centrifugation at 5,000 x g for 10 min at 4°C. The cell pellets obtained were resuspended in phosphate-buffered saline (PBS)-1 mM phenylmethylsulfonyl fluoride-20 μg of DNase I/ml and lysed by two passages through a French press. Lysates were centrifuged at 22,000 x g for 30 min. The insoluble proteins in the pellet were washed three times and resuspended in PBS containing 1% Triton X-100. Proteins were separated in sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) gels. Gel strips containing GST fusion proteins were cut, and the proteins were eluted with a Mini Trans-Blot cell (Bio-Rad, Hercules, Calif.). The resulting fusion proteins were detected by Western blotting with mouse anti-GST antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.), and their concentrations were estimated by comparison with bovine serum albumin standards in a Coomassie brilliant blue R-250 (Bio-Rad)-stained SDS-PAGE gel.
Generation of antibodies against the various GST-S fusion proteins. One milligram of each of the proteins S1, S2, S3, S9, and S10 was mixed with an equal volume of complete Freund's adjuvant (Sigma, St. Louis, Mo.) and used for the immunization of New Zealand White rabbits. Two rabbits were used to raise antibodies against each respective antigen. Three weeks after the initial immunization, the rabbits were given booster injections at 2-week intervals. Incomplete Freund's adjuvant (Sigma) was used for subsequent booster injections. Ten milliliters of blood was harvested from the rabbits each time after the 4th, 6th, 8th, 12th, 14th, and 16th injections. All procedures for the use of laboratory animals were done in accordance with the regulations and guidelines of Animal Research Ethics Committee.
Expression of recombinant S proteins in mammalian cells. Cos7 cells were used as the mammalian expression system, and the resulting S proteins were used for Western blotting, immunoprecipitation, and indirect immunofluorescence analysis. Monolayers of Cos7 cells, grown in a 21-cm2 dish were subjected to vTF7-3 vaccinia virus infection at a multiplicity of infection (MOI) of 1 PFU per cell. Transient transfections of cells with pKT-S, pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, and pKT-S16 plasmids were carried out at 1 h postinfection with Effectene transfection reagents (QIAGEN, Valencia, Calif.) according to the manufacturer's protocol as previously described (S. Shen et al., submitted for publication). For control experiments, cells were infected with the vTF7-3 vaccinia virus and mock transfected with the empty vector pKT0.
Western blot analysis. Cos7 cells were infected with 1 PFU of vTF7-3 vaccinia virus/cell for 1 h and transfected with pKT-S as described above. Cell lysates were prepared with 1x SDS loading buffer under reducing conditions (60 mM Tris-HCl [pH 6.8], 1% SDS, 20 mM dithiothreitol, 10% glycerol, 0.02% bromophenol blue). Proteins were separated in 10% PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked in 5% nonfat milk in PBS with 0.05% Tween 20 and probed with rabbit antisera (1:10,000 to 1:60,000) raised against the various S fusion proteins at 4°C overnight. The membranes were incubated with preimmune sera (1:4,000) as controls. The membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, Ill.) at a dilution of 1:2,000 for 1 h at room temperature and developed with enhanced chemiluminescence reagent (Pierce). When a SARS patient's serum (P6) was used, it was diluted 1:200 and the secondary antibody was diluted 1:2,000. For loading control, membranes were stripped with stripping buffer (2% SDS, 100 mM ?-mercaptoethanol, 62.5 mM Tris-HCl [pH 6.8]) for 30 min at 65°C and washed extensively with PBS-Tween 20 before being reprobed with a mouse anti-?-actin antibody (Sigma) at a dilution of 1:3,000.
Radiolabeled immunoprecipitation. Cos7 cells were infected with vaccinia virus vTF7-3 and transfected with pKT-S or plasmids expressing the S C-terminal deletion mutant proteins (pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, and pKT-S16) as described above. Cells were mock transfected with pKT0 as controls. At 3 h posttransfection, the cells were starved for 30 min with methionine-cysteine (Met-Cys)-free medium before being labeled with [35S]Met-Cys for 1 h and chased with 4 mM cold Met-Cys for 2 h. For the time course experiment, the chase periods before harvesting the cells were 0, 1/2, 1, 2, 4, and 6 h. For virus-infected Vero E6 cells, cells were infected at a MOI of 5, radiolabeled at 5 h postinfection for 1 h, and chased for 3 h. Cells were lysed with lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1% NP-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and centrifuged at 16,000 x g for 10 min. A portion of the supernatant (300 μl) was incubated for 1/2 h with 5 μl of antiserum, followed by a 1-h incubation with protein A-Sepharose beads (Roche Diagnostics). The beads were washed three times with lysis buffer. Twenty microliters of 1x SDS loading buffer was added to the beads and boiled for 5 min at 100°C. For the EndoH treatment, the beads were resuspended in EndoH buffer provided by the manufacturer and the immunoprecipitated proteins were eluted by boiling for 5 min. Samples were treated with EndoH enzyme (Roche Diagnostics) at 37°C for 2 h. For the control, samples were treated in the same manner except no enzyme was added. All the samples were separated in a SDS-7.5% PAGE gel and visualized by autoradiography.
Indirect immunofluorescence. Cos7 cells grown on Permanox slide (Nalge Nunc International, Naperville, Ill.) were infected and transfected as described above. At 8 h posttransfection, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and blocked with PBS containing 1% bovine serum albumin for 30 min; they were then incubated with the primary antibody (1:200) for 1.5 h, washed, and incubated with the fluorescein isothiocyanate-conjugated secondary antibody (1:200; Santa Cruz Biotechnology) for 1 h. All incubations and washes were performed at room temperature. Slides were mounted with fluorescence mounting medium (Dako Cytomation) and analyzed on an AxioVision fluorescence light microscope (Carl Zeiss Vision GmbH, Hallbergmoss, Germany).
Neutralization assay. To determine the neutralizing activities of the rabbit antisera, we performed a neutralization assay using a 96-well plate. Vero E6 cells (2 x 104) were grown in 200 μl of medium in each well of the 96-well plates and incubated at 37°C. Serial dilutions of rabbit sera with the medium in ratios of 1:10 to 1:1,280 were prepared. Diluted rabbit antisera (0.1 ml) were mixed with 0.1 ml of SARS-CoV at 200 (or 1,000) 50% tissue culture infective doses (TCID50) for 1 h at 37°C before being added into the respective wells. At each dilution, antiserum and virus mixtures were added to eight wells. The 96-well plate was incubated in a CO2 incubator for 4 days to observe the cytopathic effect. The ratios of infected wells to uninfected wells were determined under microscopy. The titers of the neutralizing antibodies were calculated by the Reed-Muench method (21) and expressed as TCID50 per 0.1 ml. All experiments were carried out in duplicate.
RESULTS
Generation of rabbit antibodies against the different regions on the S protein. Previous studies have shown that antibodies targeting bacterially expressed amino acids (aa) 548 to 567, 607 to 627 (37), and 803 to 828 (36) of S exhibit some neutralizing activities. We aim to identify the neutralizing regions that could elicit strong neutralizing antibodies. To determine the neutralizing regions on the bacterially expressed SARS-CoV S protein, we expressed five fragments of S covering the entire ectodomain (Fig. 1). Two fragments covered the S1 region (S1 and S3), two fragments covered the S2 region (S9 and S10), and one fragment covered a portion of both S1 and S2 (S2). S9 and S10 contain the HR1 and HR2 regions, respectively. Antigens for S1, S2, S3, S9, and S10 were injected into rabbits to raise polyclonal antibodies against their respective target regions. The immunizations were carried out as mentioned in Materials and Methods. Multiple booster injections were performed in an attempt to increase the titers and specificity of antibodies. The specificity and sensitivity of the antisera were determined after each bleed by Western blotting and immunoprecipitation (data not shown).
Detection of denatured form of SARS-CoV S protein by Western blotting. The sensitivities of the antibodies and their specificities for the full-length S protein, expressed in mammalian Cos7 cells, were determined by Western blot analysis. As postulated, anti-S antibodies from the serum of a patient (P6) who has recovered from SARS-CoV infection (28) could detect two major bands of full-length S protein, the 140-kDa unglycosylated form and the 200-kDa glycosylated form (Fig. 2, lane 1), in the lysates of Cos7 cells transfected with the full-length S plasmid, pKT-S. These are specific S bands, as they were not detected in mock-transfected cells (lane 2) and by serum from a healthy donor (lanes 13 and 14). We observed similar results using the antibodies that we raised against the five S fusion proteins (Fig. 2, lanes 3, 5, 7, 9, and 11). No specific bands were detected in mock-transfected cells (lanes 4, 6, 8, 10, and 12) and by preimmune sera from the same rabbits (lanes 15 to 24). The results indicate that all the antibodies raised against the various S fragments could specifically bind to S in denaturing condition by targeting different regions of the linearized S protein. Therefore, these antibodies are specific to the denatured full-length SARS-CoV S protein.
Detection of the undenatured form of S in transfected cells. To determine the specificities of the antibodies for the undenatured form of the S protein and their respective target regions on the S protein, we carried out immunoprecipitation experiments using the various antibodies. Lysates of Cos7 cells infected with vTF7-3 vaccinia virus and transfected with pKT-S and plasmids encoding the mutant proteins with C-terminal deletions, pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, and pKT-S16, were immunoprecipitated with P6 serum and the five antibodies raised against the S1, S2, S3, S9, and S10 regions. The SARS-CoV full-length S protein and the proteins with C-terminal deletions can be detected by immunoprecipitation with the P6 serum (Fig. 3f). As expected, the sizes of the S proteins with deletions in the C terminus decrease from S11 to S16, as more amino acid residues were removed from the C terminus. Two forms of S (200 and 210 kDa) were detected in cell lysates transfected with the full-length S construct (Fig. 3a to f, lanes 1). These might represent the mature and immature forms of the full-length S protein. Antibodies targeting regions S1, S2, and S3 individually can detect the full-length S and all the proteins with C-terminal deletions (Fig. 3a to c). Antibodies targeting region S9 can detect full-length S and S11 to S15 proteins. No specific band was detected in the S16 lane because S16 does not contain the S9 region (Fig. 3d). Antibodies targeting S10 could only detect the protein bands expressed by pKT-S and pKT-S11 to pKT-S13 (Fig. 3e). The regions expressed by pKT-S14, pKT-S15, and pKT-S16 do not include the S10 region and thus rabbit anti-S10 does not detect any band in these lanes, showing that these antibodies are highly specific to their target regions. The conformation of the S protein was retained in the nondenaturing lysis buffer in immunoprecipitation, and the results showed that the protein possesses an epitope(s) which can be recognized by all five antibodies raised against the five fusion proteins. The 140-kDa unglycosylated form of S was not detected by immunoprecipitation with any of the antibodies used. The results suggested that either the unglycosylated S protein was not recognized by these antibodies under nondenaturing conditions or the 140-kDa form of S did not accumulate in pulse-chase experiments.
Detection of the native S protein in virus-infected cells and cultured media. To further investigate whether these polyclonal antibodies can specifically bind to the native S protein, the S proteins synthesized in virus-infected cells and released as components of the virion to cultured medium were analyzed in an immunoprecipitation assay using the lysis buffer described above. As shown in Fig. 4a, a specific S protein was detected in both cell lysates (top, lanes 2, 6, and 10; bottom, lanes 2 and 6) and media (top, lanes 4, 8, and 12; bottom, lanes 4 and 8). The S protein was not detected in cells and media of mock-infected cells (lanes 1, 3, 5, 7, 9, and 11) or in infected cells and medium using preimmune serum of a rabbit immunized with the S10 peptide (immunoprecipitations using other preimmune sera were negative and are not shown). It was noted that the S species in media is slightly larger than majority of S species in cell lysates, suggesting (i) that the S protein had matured from 200 to 210 kDa and (ii) that the mature and modified S protein of 210 kDa was assembled into a virion. The results confirmed that these antibodies are specific for the native S protein in virus-infected cells and virus particles.
Maturation of the 200-kDa S protein to the 210-kDa EndoH-resistant form. Two S-specific bands were observed in the immunoprecipitation experiments. We suspect that the 210-kDa band was the mature glycosylated S protein and that the 200-kDa band is the immature glycosylated S protein. To investigate the maturation of S, we carried out a pulse-chase labeling experiment. Cos7 cells infected with vaccinia virus vTF7-3 and transfected with pKT-S were radiolabeled with [35S]Met-Cys and chased with cold Met-Cys for 1/2, 1, 2, 4, and 6 h, followed by immunoprecipitation with rabbit anti-S10. The results showed a gradual increase in the 210-kDa band accompanied by a gradual decrease in the 200-kDa band (Fig. 4b, lanes 1 to 8). The yields of 200- and 210-kDa proteins decreased after chasing for 4 h due to cell death caused by vTF7.3 infection; however, the ratio of 210- to 200-kDa protein increased. Immunoprecipitation was performed with all the other antisera (rabbit anti-S1, anti-S2, anti-S3, and anti-S9), and the same results were obtained (data not shown). To provide more evidence to support our findings, an EndoH treatment experiment was carried out to find out whether the bands were sensitive to EndoH. The radiolabeled cell lysates described above were immunoprecipitated with rabbit anti-S10 and treated or mock treated with EndoH enzyme. Results showed that the 210-kDa band was EndoH resistant and that the 200-kDa band was EndoH sensitive (Fig. 4b, lanes 9 and 10). Hence, the results demonstrated the maturation of the 200-kDa band to the 210-kDa band. Other antibodies (rabbit anti-S1, anti-S2, anti-S3, and anti-S9) can also recognize both the mature and immature forms of the S glycoprotein (data not shown).
Detection of SARS-CoV S protein on surfaces of Cos7 cells. To provide further evidence that the antibodies raised against the five fusion proteins were able to recognize the S protein, we performed indirect immunofluorescence experiments under nonpermeabilized conditions. After infection with the vTF7-3 vaccinia virus and transfection with pKT-S, sufficient time (8 h posttransfection) was allowed for the S protein to be expressed and transported to the cell surface. The mature S protein could exist in trimeric or oligomeric form on the cell surface, and these could be clearly detected with any of the antibodies raised against the various S proteins (Fig. 5, left). The binding of the antibodies against S1, S2, S3, S9, and S10 to the S protein on the cell surface suggests that these antibodies might recognize the properly folded, mature form of the SARS-CoV S protein, which was expressed and transported to the cell surface. The experiment was repeated three times, and all the results were consistent.
A region in S2 can elicit neutralizing activity. After determining that all the antibodies could recognize the native form of the SARS CoV S protein, we tested their neutralizing activities. All antisera from rabbits injected with the various S proteins were tested for their neutralizing activities after each bleed. Results for rabbits injected with bacterially expressed S1, S2, S3, and S9 showed negative response. Sera from rabbits injected with S10 showed neutralizing activities after the fourth injection but not the preimmune sera from the same rabbits. Initial tests using SARS-CoV at 200 TCID50/well showed high titers (1:364) of neutralizing antibodies in all the anti-S10 antisera, beginning with serum bled after the 8th injection (Table 2). The 16th injection was the last booster injection, and the rabbit was sacrificed at this stage. The neutralizing titer was comparable to, if not higher than, the titers detected in SARS patients. The antibody response in SARS patients at 100 TCID50/well ranges from 1:150 to 1:475 over a period of 210 days (23). To ascertain the results above, serum samples after the 4th, 6th, 8th, 12th, 14th, and 16th injections with S10 were used for neutralizing tests using SARS-CoV at 1,000 TCID50/well (Table 3). Neutralizing activities were found to be as high as 1:189.2 at l,000 TCID50/well. This result showed that the S10 fragments expressed in E. coli covering amino acid residues 1055 to 1192 can stimulate neutralizing antibodies but that those fragments covering all other regions of the ectodomain of S cannot. It was interesting that S10 (aa 1055 to 1192), which contained the HR2 domain, could elicit a strong neutralizing response but not S9 (aa 798 to 1055), which covered the HR1 domain. S1, S2, S3, and S9 could possess neutralizing epitopes that are conformation or glycosylation dependent and could have their epitopes impaired in the bacterial expression system, which explains their lack of neutralizing activity.
DISCUSSION
The S protein of CoV is an important determinant of tissue tropism, as it binds to cellular receptors on the host cell and as it also mediates virus and cellular membrane fusion. For SARS-CoV, it appears that neutralizing antibodies against S can provide some form of immunity against SARS-CoV infection (2, 5, 6, 12, 27). In this study, we raised rabbit polyclonal antibodies against five bacterially expressed S fragments, covering the entire ectodomain (aa 48 to 1192). As these proteins were expressed in E. coli, the antibodies might not be able to recognize some conformation- or glycosylation-dependent neutralizing epitopes on S. Our analysis is also likely to be limited to linear epitopes since the proteins were extracted from SDS denaturing gels and eluted in the presence of SDS, although we cannot rule out the possibility that the protein can maintain some degree of folding. Interestingly, we found that the antibodies not only were able to recognize denatured S proteins (expressed in mammalian cells) in Western blot analysis but also could immunoprecipitate native S proteins from cell lysates (or media) of transfected and virus-infected cells (Fig. 2 and 4a). The results indicate that these antibodies bind to linear and exposed epitopes present in the folded S and/or to flexible regions, such as turns, that exist in the structure of S. Importantly, immunoprecipitation experiments showed that these antibodies could bind to both the EndoH-sensitive, glycosylated (200-kDa) full-length S and the EndoH-resistant glycosylated S (210 kDa). This is also supported by indirect immunofluorescence experiments which showed that these antibodies could recognize S protein expressed on the cell surface. It is known that the glycosylated S protein has to be transported to the Golgi apparatus before it undergoes maturation and that it is then transported to the cell surface (19, 31). We found that maturation of the S protein of SARS-CoV results in an increase in apparent molecular mass from 200 to 210 kDa. It is also shown that the 210-kDa fully glycoslyated form of SARS CoV S is later incorporated into virions which are released into the medium of SARS-CoV-infected Vero E6 culture (Fig. 4a).
Neutralization assays showed that antibodies raised against GST-S10 were capable of neutralizing SARS-CoV replication in Vero E6 cells at a titer of up to 1:364 at 200 TCID50, which is comparable to the level obtained for convalescent patients (23). Analysis of sera taken after accumulative immunizations displayed a steady increase in neutralizing titer, indicating that the immunized rabbits showed a specific immune response to GST-S10. None of the other antigens was capable of inducing neutralizing antibodies, which could indicate that there was an absence of neutralizing epitopes in aa 48 to 1055 of the S protein. However, this phenomenon is most unlikely, as a human antibody against aa 261 to 672 has been shown to have neutralizing properties (27) (see below for a discussion of the receptor binding domain). The more probable explanation is that neutralizing epitopes in this region are heavily glycoslyated and/or contain rigid tertiary structures. Therefore, our results showed that aa 48 to 1055 of the S protein are not suitable for use in the development of vaccines that are peptide based or based on proteins expressed in nonmammalian systems that are not able to mimic the glycoslyation process or proper folding in mammalian cells.
The first step in CoV infection is the attachment of virions to host cells, and, for SARS-CoV, ACE-2 has been identified as the cellular receptor that binds to the SARS-CoV S protein (17). A domain in the N terminus of the S protein, approximately aa 300 to 510, is the receptor binding domain (1, 32, 33). Subsequently, the fusion of the lipid bilayer of the viral envelope with the host cell membrane is mediated by an internal fusion peptide of S (4, 10). The CoV S protein is a class I virus fusion protein and contains two heptad repeat domains (HR1 and HR2) in S2 or the C-terminal region. These domains are postulated to play crucial roles in defining the oligomeric structure of S and hence facilitate the fusion between viral and cellular membranes (10). For SARS-CoV, HR2 is located close to the transmembrane anchor (aa 1195 to 1223) and HR1 is 170 aa upstream of it (aa 891 to 975) (13). Interestingly, S10 (aa 1029 to 1192) encompasses the HR2 region, and our results are consistent with the findings of several other laboratories that peptides from the HR2 region can block SARS-CoV infection (4, 35, 38). A peptide containing HR2 can potentially be developed as a therapeutic reagent for treatment of SARS-CoV infection (4). We found that a peptide containing HR2 (S10) is a potential vaccine candidate and would be suitable for subunit vaccine development.
Biochemical studies have shown that peptides corresponding to the HR1 and HR2 of the SARS-CoV S protein can associate into an antiparallel six-helix bundle with structural features typical of those of the other known class I fusion proteins, suggesting that the membrane fusion and cell entry mechanisms exploited by SARS-CoV are similar to those for other CoVs, such as mouse hepatitis virus (3, 13, 18, 30, 35, 38). In the full-length S protein, the HR1-HR2 structure brings the fusion peptide, predicted to be near the N terminus of HR1 (4), into close proximity to the transmembrane domain, and this facilitates the fusion between viral and cellular membranes, allowing the virus to enter the cell. It is very probable that, by binding to the HR2 domain with high affinity, anti-S10 antibodies could block the interaction between HR1 and HR2 and hence prevent SARS-CoV fusion with the host cells.
As bacterially expressed proteins would be easy and cost-effective to produce on a large scale, the S10 fragment (aa 1029 to 1192) identified in this study may be an ideal vaccine candidate for SARS-CoV. In future studies, it will be critical to determine if the immunization of peptide S10 can prevent SARS-CoV replication and, more importantly, prevent disease in animal models. It is also important to delineate the precise contribution of this S region to membrane fusion. We are currently mapping monoclonal antibodies obtained from mice that have been immunized with GST-S10 proteins and determining their binding and neutralizing properties. Last, the recombinant S10 protein and anti-S10 antibodies can potentially be developed for antiviral treatments, as it has recently been shown to be possible to control SARS-CoV infection in ferrets with a neutralizing antibody directed against S (29).
ACKNOWLEDGMENTS
We thank Ng Yi Zhen, Daphne Chan, Tham Puay Yoke, Choi Yook-Wah, Hong He, Fang Zhizheng, and Li Pingping for technical assistance.
This work was supported by a grant from the Agency for Science, Technology and Research, Singapore.
C.-T.K. and A.Z. contributed equally to this article.
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Wuhan Institute of Biological Products, Wuhan, People's Republic of China
ABSTRACT
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
INTRODUCTION
A novel coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS) (8, 9, 15, 20). CoVs are positive-strand RNA viruses, and the virion consists of a nucleocapsid (N) core surrounded by an envelope containing three membrane proteins, spike (S), membrane (M), and envelope (E), that are common to all members of the genus (for reviews, see references 16 and 24). The S protein, which forms morphologically characteristic projections on the virion surface, binds to host receptors and mediates membrane fusion. The M and E proteins are important for viral particle assembly, while N is important for viral RNA packaging.
The S protein of CoV is a type 1 integral membrane glycoprotein. It is cotranslationally glycosylated and oligomerized at the endoplasmic reticulum. Its N-linked high-mannose side chains are trimmed and modified and become endoglycosidase H (EndoH) resistant during the transportation to the Golgi apparatus. For some but not all CoVs, the S protein is cleaved into the N-terminal S1 and C-terminal S2 subunits, which contain receptor binding and membrane fusion domains (10, 32), respectively. The mature forms of S are assembled into virions, which release from infected cells. A portion of S is transported to the plasma membrane, resulting in cell-cell fusion or formation of syncytia. The S protein belongs to the class 1 viral fusion proteins and contains two heptad repeat domains (HR1 and HR2) in S2 or the C-terminal region. These two domains interact and play a crucial role in mediating virus-cell membrane fusion and entry of virus into cells.
The S protein of CoV is known to be responsible for inducing host immune responses and virus neutralization by antibodies (5, 7, 14, 25). For SARS-CoV, it has been demonstrated that prior infection provided protective immunity in a mouse model and that the passive transfer of neutralizing antibodies to naive mice also protected them from infection (26). This suggests that neutralizing antibodies have an important role in reducing the viral load or preventing viral replication. A DNA vaccine encoding the S protein alone induces T-cell and neutralizing antibody responses and protected mice from SARS-CoV infection (34), suggesting that the S protein is indeed the primary target for viral neutralization in SARS-CoV infection. This finding was also confirmed by at least four independent studies that use a carrier virus to express S in mice or primates (2, 5, 6, 11). These reports highlight the potential for developing peptide-based vaccine candidates if neutralizing epitopes of S could be identified.
In this study, we aim to identify neutralizing epitopes in the S protein of SARS-CoV, which may be used for the development of vaccine or therapeutic agents against SARS-CoV infection. We expressed different regions of S as glutathione S-transferase (GST) fusion proteins and used them to raise antibodies in rabbits. These polyclonal antibodies were then tested for their specificities for the native and denatured forms of S in Western blot, immunoprecipitation, and immunofluorescence analyses as well as for their capacities to inhibit SARS-CoV replication in Vero E6 culture. A novel neutralizing epitope in S2 containing HR2 was identified.
MATERIALS AND METHODS
Cells and viruses. The monkey kidney cell lines Cos7 and Vero E6, used in this study, were purchased from the American Type Culture Collection (Manassas, Va.). Cos7 and Vero E6 cells were cultured at 37°C in a 5% CO2 incubator in Dulbecco modified Eagle medium containing 0.1 mg of streptomycin/ml, 100 U of penicillin/ml, and 10% fetal bovine serum (HyClone, Logan, Utah). SARS-CoV viral strain Sin2774 (GenBank accession no. AY283798) (22) and recombinant vaccinia virus vTF7-3 expressing bacteriophage T7 RNA polymerase were grown in Vero E6 cells.
Construction of plasmids. (i) Plasmids for expression in mammalian cells. Viral RNA of Singapore strain Sin2774 was extracted and used for amplification of the S gene by reverse transcription-PCR (RT-PCR) with specific primers. The PCR products were digested with BamHI and StuI and ligated into BamHI/EcoRV-cut pKT0, resulting in plasmid pKT-S under the control of a T7 promoter. Specific primers were designed to amplify a strain Sin2774 sequence from nucleotide positions 21476 to 25171, 25066, 24934, 24415, 24157, and 23866. The six RT-PCR products were digested with BamHI and StuI and ligated to BamHI/EcoRV-cut pKT0 under the control of a T7 promoter, giving rise to plasmids pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, pKT-S16, respectively. These constructs encode the C-terminally truncated S proteins, as shown in Fig. 1.
(ii) Plasmids for expression in Escherichia coli. Five PCR fragments encoding the S1, S2, S3, S9, and S10 fragments (Fig. 1) were obtained with the primers listed in Table 1. All primers used were purchased from Research Biolabs or Proligo Pte Ltd. (Singapore). The PCR products were digested by BamHI/XhoI and ligated into the BamHI/XhoI-cut pGEX4T1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden) to obtain plasmids pGEX-S1, pGEX-S2, pGEX-S3, pGEX-S9, and pGEX-S10 for the expression of GST fusion proteins (Fig. 1). The GST tag was located at the N terminus of the fusion protein.
Purification of the recombinant S proteins expressed in E. coli. Plasmids pGEX-S1, pGEX-S2, pGEX-S3, pGEX-S9, and pGEX-S10 were separately transformed into BL21(DE3) cells. A single colony from each plate was grown at 37°C overnight in Luria-Bertani (LB) medium containing ampicillin (100 μg/ml). Five milliliters of the resulting cultures was inoculated into 2 liters of LB medium containing ampicillin (100 μg/ml), which was incubated in a shaker at 37°C until the optical density at 600 nm reached 0.6. Expression of proteins was induced with 1 mM IPTG (isopropyl-?-D-thiogalactopyranoside). Cells were harvested 2 h after induction by centrifugation at 5,000 x g for 10 min at 4°C. The cell pellets obtained were resuspended in phosphate-buffered saline (PBS)-1 mM phenylmethylsulfonyl fluoride-20 μg of DNase I/ml and lysed by two passages through a French press. Lysates were centrifuged at 22,000 x g for 30 min. The insoluble proteins in the pellet were washed three times and resuspended in PBS containing 1% Triton X-100. Proteins were separated in sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) gels. Gel strips containing GST fusion proteins were cut, and the proteins were eluted with a Mini Trans-Blot cell (Bio-Rad, Hercules, Calif.). The resulting fusion proteins were detected by Western blotting with mouse anti-GST antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.), and their concentrations were estimated by comparison with bovine serum albumin standards in a Coomassie brilliant blue R-250 (Bio-Rad)-stained SDS-PAGE gel.
Generation of antibodies against the various GST-S fusion proteins. One milligram of each of the proteins S1, S2, S3, S9, and S10 was mixed with an equal volume of complete Freund's adjuvant (Sigma, St. Louis, Mo.) and used for the immunization of New Zealand White rabbits. Two rabbits were used to raise antibodies against each respective antigen. Three weeks after the initial immunization, the rabbits were given booster injections at 2-week intervals. Incomplete Freund's adjuvant (Sigma) was used for subsequent booster injections. Ten milliliters of blood was harvested from the rabbits each time after the 4th, 6th, 8th, 12th, 14th, and 16th injections. All procedures for the use of laboratory animals were done in accordance with the regulations and guidelines of Animal Research Ethics Committee.
Expression of recombinant S proteins in mammalian cells. Cos7 cells were used as the mammalian expression system, and the resulting S proteins were used for Western blotting, immunoprecipitation, and indirect immunofluorescence analysis. Monolayers of Cos7 cells, grown in a 21-cm2 dish were subjected to vTF7-3 vaccinia virus infection at a multiplicity of infection (MOI) of 1 PFU per cell. Transient transfections of cells with pKT-S, pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, and pKT-S16 plasmids were carried out at 1 h postinfection with Effectene transfection reagents (QIAGEN, Valencia, Calif.) according to the manufacturer's protocol as previously described (S. Shen et al., submitted for publication). For control experiments, cells were infected with the vTF7-3 vaccinia virus and mock transfected with the empty vector pKT0.
Western blot analysis. Cos7 cells were infected with 1 PFU of vTF7-3 vaccinia virus/cell for 1 h and transfected with pKT-S as described above. Cell lysates were prepared with 1x SDS loading buffer under reducing conditions (60 mM Tris-HCl [pH 6.8], 1% SDS, 20 mM dithiothreitol, 10% glycerol, 0.02% bromophenol blue). Proteins were separated in 10% PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked in 5% nonfat milk in PBS with 0.05% Tween 20 and probed with rabbit antisera (1:10,000 to 1:60,000) raised against the various S fusion proteins at 4°C overnight. The membranes were incubated with preimmune sera (1:4,000) as controls. The membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, Ill.) at a dilution of 1:2,000 for 1 h at room temperature and developed with enhanced chemiluminescence reagent (Pierce). When a SARS patient's serum (P6) was used, it was diluted 1:200 and the secondary antibody was diluted 1:2,000. For loading control, membranes were stripped with stripping buffer (2% SDS, 100 mM ?-mercaptoethanol, 62.5 mM Tris-HCl [pH 6.8]) for 30 min at 65°C and washed extensively with PBS-Tween 20 before being reprobed with a mouse anti-?-actin antibody (Sigma) at a dilution of 1:3,000.
Radiolabeled immunoprecipitation. Cos7 cells were infected with vaccinia virus vTF7-3 and transfected with pKT-S or plasmids expressing the S C-terminal deletion mutant proteins (pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, and pKT-S16) as described above. Cells were mock transfected with pKT0 as controls. At 3 h posttransfection, the cells were starved for 30 min with methionine-cysteine (Met-Cys)-free medium before being labeled with [35S]Met-Cys for 1 h and chased with 4 mM cold Met-Cys for 2 h. For the time course experiment, the chase periods before harvesting the cells were 0, 1/2, 1, 2, 4, and 6 h. For virus-infected Vero E6 cells, cells were infected at a MOI of 5, radiolabeled at 5 h postinfection for 1 h, and chased for 3 h. Cells were lysed with lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1% NP-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and centrifuged at 16,000 x g for 10 min. A portion of the supernatant (300 μl) was incubated for 1/2 h with 5 μl of antiserum, followed by a 1-h incubation with protein A-Sepharose beads (Roche Diagnostics). The beads were washed three times with lysis buffer. Twenty microliters of 1x SDS loading buffer was added to the beads and boiled for 5 min at 100°C. For the EndoH treatment, the beads were resuspended in EndoH buffer provided by the manufacturer and the immunoprecipitated proteins were eluted by boiling for 5 min. Samples were treated with EndoH enzyme (Roche Diagnostics) at 37°C for 2 h. For the control, samples were treated in the same manner except no enzyme was added. All the samples were separated in a SDS-7.5% PAGE gel and visualized by autoradiography.
Indirect immunofluorescence. Cos7 cells grown on Permanox slide (Nalge Nunc International, Naperville, Ill.) were infected and transfected as described above. At 8 h posttransfection, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and blocked with PBS containing 1% bovine serum albumin for 30 min; they were then incubated with the primary antibody (1:200) for 1.5 h, washed, and incubated with the fluorescein isothiocyanate-conjugated secondary antibody (1:200; Santa Cruz Biotechnology) for 1 h. All incubations and washes were performed at room temperature. Slides were mounted with fluorescence mounting medium (Dako Cytomation) and analyzed on an AxioVision fluorescence light microscope (Carl Zeiss Vision GmbH, Hallbergmoss, Germany).
Neutralization assay. To determine the neutralizing activities of the rabbit antisera, we performed a neutralization assay using a 96-well plate. Vero E6 cells (2 x 104) were grown in 200 μl of medium in each well of the 96-well plates and incubated at 37°C. Serial dilutions of rabbit sera with the medium in ratios of 1:10 to 1:1,280 were prepared. Diluted rabbit antisera (0.1 ml) were mixed with 0.1 ml of SARS-CoV at 200 (or 1,000) 50% tissue culture infective doses (TCID50) for 1 h at 37°C before being added into the respective wells. At each dilution, antiserum and virus mixtures were added to eight wells. The 96-well plate was incubated in a CO2 incubator for 4 days to observe the cytopathic effect. The ratios of infected wells to uninfected wells were determined under microscopy. The titers of the neutralizing antibodies were calculated by the Reed-Muench method (21) and expressed as TCID50 per 0.1 ml. All experiments were carried out in duplicate.
RESULTS
Generation of rabbit antibodies against the different regions on the S protein. Previous studies have shown that antibodies targeting bacterially expressed amino acids (aa) 548 to 567, 607 to 627 (37), and 803 to 828 (36) of S exhibit some neutralizing activities. We aim to identify the neutralizing regions that could elicit strong neutralizing antibodies. To determine the neutralizing regions on the bacterially expressed SARS-CoV S protein, we expressed five fragments of S covering the entire ectodomain (Fig. 1). Two fragments covered the S1 region (S1 and S3), two fragments covered the S2 region (S9 and S10), and one fragment covered a portion of both S1 and S2 (S2). S9 and S10 contain the HR1 and HR2 regions, respectively. Antigens for S1, S2, S3, S9, and S10 were injected into rabbits to raise polyclonal antibodies against their respective target regions. The immunizations were carried out as mentioned in Materials and Methods. Multiple booster injections were performed in an attempt to increase the titers and specificity of antibodies. The specificity and sensitivity of the antisera were determined after each bleed by Western blotting and immunoprecipitation (data not shown).
Detection of denatured form of SARS-CoV S protein by Western blotting. The sensitivities of the antibodies and their specificities for the full-length S protein, expressed in mammalian Cos7 cells, were determined by Western blot analysis. As postulated, anti-S antibodies from the serum of a patient (P6) who has recovered from SARS-CoV infection (28) could detect two major bands of full-length S protein, the 140-kDa unglycosylated form and the 200-kDa glycosylated form (Fig. 2, lane 1), in the lysates of Cos7 cells transfected with the full-length S plasmid, pKT-S. These are specific S bands, as they were not detected in mock-transfected cells (lane 2) and by serum from a healthy donor (lanes 13 and 14). We observed similar results using the antibodies that we raised against the five S fusion proteins (Fig. 2, lanes 3, 5, 7, 9, and 11). No specific bands were detected in mock-transfected cells (lanes 4, 6, 8, 10, and 12) and by preimmune sera from the same rabbits (lanes 15 to 24). The results indicate that all the antibodies raised against the various S fragments could specifically bind to S in denaturing condition by targeting different regions of the linearized S protein. Therefore, these antibodies are specific to the denatured full-length SARS-CoV S protein.
Detection of the undenatured form of S in transfected cells. To determine the specificities of the antibodies for the undenatured form of the S protein and their respective target regions on the S protein, we carried out immunoprecipitation experiments using the various antibodies. Lysates of Cos7 cells infected with vTF7-3 vaccinia virus and transfected with pKT-S and plasmids encoding the mutant proteins with C-terminal deletions, pKT-S11, pKT-S12, pKT-S13, pKT-S14, pKT-S15, and pKT-S16, were immunoprecipitated with P6 serum and the five antibodies raised against the S1, S2, S3, S9, and S10 regions. The SARS-CoV full-length S protein and the proteins with C-terminal deletions can be detected by immunoprecipitation with the P6 serum (Fig. 3f). As expected, the sizes of the S proteins with deletions in the C terminus decrease from S11 to S16, as more amino acid residues were removed from the C terminus. Two forms of S (200 and 210 kDa) were detected in cell lysates transfected with the full-length S construct (Fig. 3a to f, lanes 1). These might represent the mature and immature forms of the full-length S protein. Antibodies targeting regions S1, S2, and S3 individually can detect the full-length S and all the proteins with C-terminal deletions (Fig. 3a to c). Antibodies targeting region S9 can detect full-length S and S11 to S15 proteins. No specific band was detected in the S16 lane because S16 does not contain the S9 region (Fig. 3d). Antibodies targeting S10 could only detect the protein bands expressed by pKT-S and pKT-S11 to pKT-S13 (Fig. 3e). The regions expressed by pKT-S14, pKT-S15, and pKT-S16 do not include the S10 region and thus rabbit anti-S10 does not detect any band in these lanes, showing that these antibodies are highly specific to their target regions. The conformation of the S protein was retained in the nondenaturing lysis buffer in immunoprecipitation, and the results showed that the protein possesses an epitope(s) which can be recognized by all five antibodies raised against the five fusion proteins. The 140-kDa unglycosylated form of S was not detected by immunoprecipitation with any of the antibodies used. The results suggested that either the unglycosylated S protein was not recognized by these antibodies under nondenaturing conditions or the 140-kDa form of S did not accumulate in pulse-chase experiments.
Detection of the native S protein in virus-infected cells and cultured media. To further investigate whether these polyclonal antibodies can specifically bind to the native S protein, the S proteins synthesized in virus-infected cells and released as components of the virion to cultured medium were analyzed in an immunoprecipitation assay using the lysis buffer described above. As shown in Fig. 4a, a specific S protein was detected in both cell lysates (top, lanes 2, 6, and 10; bottom, lanes 2 and 6) and media (top, lanes 4, 8, and 12; bottom, lanes 4 and 8). The S protein was not detected in cells and media of mock-infected cells (lanes 1, 3, 5, 7, 9, and 11) or in infected cells and medium using preimmune serum of a rabbit immunized with the S10 peptide (immunoprecipitations using other preimmune sera were negative and are not shown). It was noted that the S species in media is slightly larger than majority of S species in cell lysates, suggesting (i) that the S protein had matured from 200 to 210 kDa and (ii) that the mature and modified S protein of 210 kDa was assembled into a virion. The results confirmed that these antibodies are specific for the native S protein in virus-infected cells and virus particles.
Maturation of the 200-kDa S protein to the 210-kDa EndoH-resistant form. Two S-specific bands were observed in the immunoprecipitation experiments. We suspect that the 210-kDa band was the mature glycosylated S protein and that the 200-kDa band is the immature glycosylated S protein. To investigate the maturation of S, we carried out a pulse-chase labeling experiment. Cos7 cells infected with vaccinia virus vTF7-3 and transfected with pKT-S were radiolabeled with [35S]Met-Cys and chased with cold Met-Cys for 1/2, 1, 2, 4, and 6 h, followed by immunoprecipitation with rabbit anti-S10. The results showed a gradual increase in the 210-kDa band accompanied by a gradual decrease in the 200-kDa band (Fig. 4b, lanes 1 to 8). The yields of 200- and 210-kDa proteins decreased after chasing for 4 h due to cell death caused by vTF7.3 infection; however, the ratio of 210- to 200-kDa protein increased. Immunoprecipitation was performed with all the other antisera (rabbit anti-S1, anti-S2, anti-S3, and anti-S9), and the same results were obtained (data not shown). To provide more evidence to support our findings, an EndoH treatment experiment was carried out to find out whether the bands were sensitive to EndoH. The radiolabeled cell lysates described above were immunoprecipitated with rabbit anti-S10 and treated or mock treated with EndoH enzyme. Results showed that the 210-kDa band was EndoH resistant and that the 200-kDa band was EndoH sensitive (Fig. 4b, lanes 9 and 10). Hence, the results demonstrated the maturation of the 200-kDa band to the 210-kDa band. Other antibodies (rabbit anti-S1, anti-S2, anti-S3, and anti-S9) can also recognize both the mature and immature forms of the S glycoprotein (data not shown).
Detection of SARS-CoV S protein on surfaces of Cos7 cells. To provide further evidence that the antibodies raised against the five fusion proteins were able to recognize the S protein, we performed indirect immunofluorescence experiments under nonpermeabilized conditions. After infection with the vTF7-3 vaccinia virus and transfection with pKT-S, sufficient time (8 h posttransfection) was allowed for the S protein to be expressed and transported to the cell surface. The mature S protein could exist in trimeric or oligomeric form on the cell surface, and these could be clearly detected with any of the antibodies raised against the various S proteins (Fig. 5, left). The binding of the antibodies against S1, S2, S3, S9, and S10 to the S protein on the cell surface suggests that these antibodies might recognize the properly folded, mature form of the SARS-CoV S protein, which was expressed and transported to the cell surface. The experiment was repeated three times, and all the results were consistent.
A region in S2 can elicit neutralizing activity. After determining that all the antibodies could recognize the native form of the SARS CoV S protein, we tested their neutralizing activities. All antisera from rabbits injected with the various S proteins were tested for their neutralizing activities after each bleed. Results for rabbits injected with bacterially expressed S1, S2, S3, and S9 showed negative response. Sera from rabbits injected with S10 showed neutralizing activities after the fourth injection but not the preimmune sera from the same rabbits. Initial tests using SARS-CoV at 200 TCID50/well showed high titers (1:364) of neutralizing antibodies in all the anti-S10 antisera, beginning with serum bled after the 8th injection (Table 2). The 16th injection was the last booster injection, and the rabbit was sacrificed at this stage. The neutralizing titer was comparable to, if not higher than, the titers detected in SARS patients. The antibody response in SARS patients at 100 TCID50/well ranges from 1:150 to 1:475 over a period of 210 days (23). To ascertain the results above, serum samples after the 4th, 6th, 8th, 12th, 14th, and 16th injections with S10 were used for neutralizing tests using SARS-CoV at 1,000 TCID50/well (Table 3). Neutralizing activities were found to be as high as 1:189.2 at l,000 TCID50/well. This result showed that the S10 fragments expressed in E. coli covering amino acid residues 1055 to 1192 can stimulate neutralizing antibodies but that those fragments covering all other regions of the ectodomain of S cannot. It was interesting that S10 (aa 1055 to 1192), which contained the HR2 domain, could elicit a strong neutralizing response but not S9 (aa 798 to 1055), which covered the HR1 domain. S1, S2, S3, and S9 could possess neutralizing epitopes that are conformation or glycosylation dependent and could have their epitopes impaired in the bacterial expression system, which explains their lack of neutralizing activity.
DISCUSSION
The S protein of CoV is an important determinant of tissue tropism, as it binds to cellular receptors on the host cell and as it also mediates virus and cellular membrane fusion. For SARS-CoV, it appears that neutralizing antibodies against S can provide some form of immunity against SARS-CoV infection (2, 5, 6, 12, 27). In this study, we raised rabbit polyclonal antibodies against five bacterially expressed S fragments, covering the entire ectodomain (aa 48 to 1192). As these proteins were expressed in E. coli, the antibodies might not be able to recognize some conformation- or glycosylation-dependent neutralizing epitopes on S. Our analysis is also likely to be limited to linear epitopes since the proteins were extracted from SDS denaturing gels and eluted in the presence of SDS, although we cannot rule out the possibility that the protein can maintain some degree of folding. Interestingly, we found that the antibodies not only were able to recognize denatured S proteins (expressed in mammalian cells) in Western blot analysis but also could immunoprecipitate native S proteins from cell lysates (or media) of transfected and virus-infected cells (Fig. 2 and 4a). The results indicate that these antibodies bind to linear and exposed epitopes present in the folded S and/or to flexible regions, such as turns, that exist in the structure of S. Importantly, immunoprecipitation experiments showed that these antibodies could bind to both the EndoH-sensitive, glycosylated (200-kDa) full-length S and the EndoH-resistant glycosylated S (210 kDa). This is also supported by indirect immunofluorescence experiments which showed that these antibodies could recognize S protein expressed on the cell surface. It is known that the glycosylated S protein has to be transported to the Golgi apparatus before it undergoes maturation and that it is then transported to the cell surface (19, 31). We found that maturation of the S protein of SARS-CoV results in an increase in apparent molecular mass from 200 to 210 kDa. It is also shown that the 210-kDa fully glycoslyated form of SARS CoV S is later incorporated into virions which are released into the medium of SARS-CoV-infected Vero E6 culture (Fig. 4a).
Neutralization assays showed that antibodies raised against GST-S10 were capable of neutralizing SARS-CoV replication in Vero E6 cells at a titer of up to 1:364 at 200 TCID50, which is comparable to the level obtained for convalescent patients (23). Analysis of sera taken after accumulative immunizations displayed a steady increase in neutralizing titer, indicating that the immunized rabbits showed a specific immune response to GST-S10. None of the other antigens was capable of inducing neutralizing antibodies, which could indicate that there was an absence of neutralizing epitopes in aa 48 to 1055 of the S protein. However, this phenomenon is most unlikely, as a human antibody against aa 261 to 672 has been shown to have neutralizing properties (27) (see below for a discussion of the receptor binding domain). The more probable explanation is that neutralizing epitopes in this region are heavily glycoslyated and/or contain rigid tertiary structures. Therefore, our results showed that aa 48 to 1055 of the S protein are not suitable for use in the development of vaccines that are peptide based or based on proteins expressed in nonmammalian systems that are not able to mimic the glycoslyation process or proper folding in mammalian cells.
The first step in CoV infection is the attachment of virions to host cells, and, for SARS-CoV, ACE-2 has been identified as the cellular receptor that binds to the SARS-CoV S protein (17). A domain in the N terminus of the S protein, approximately aa 300 to 510, is the receptor binding domain (1, 32, 33). Subsequently, the fusion of the lipid bilayer of the viral envelope with the host cell membrane is mediated by an internal fusion peptide of S (4, 10). The CoV S protein is a class I virus fusion protein and contains two heptad repeat domains (HR1 and HR2) in S2 or the C-terminal region. These domains are postulated to play crucial roles in defining the oligomeric structure of S and hence facilitate the fusion between viral and cellular membranes (10). For SARS-CoV, HR2 is located close to the transmembrane anchor (aa 1195 to 1223) and HR1 is 170 aa upstream of it (aa 891 to 975) (13). Interestingly, S10 (aa 1029 to 1192) encompasses the HR2 region, and our results are consistent with the findings of several other laboratories that peptides from the HR2 region can block SARS-CoV infection (4, 35, 38). A peptide containing HR2 can potentially be developed as a therapeutic reagent for treatment of SARS-CoV infection (4). We found that a peptide containing HR2 (S10) is a potential vaccine candidate and would be suitable for subunit vaccine development.
Biochemical studies have shown that peptides corresponding to the HR1 and HR2 of the SARS-CoV S protein can associate into an antiparallel six-helix bundle with structural features typical of those of the other known class I fusion proteins, suggesting that the membrane fusion and cell entry mechanisms exploited by SARS-CoV are similar to those for other CoVs, such as mouse hepatitis virus (3, 13, 18, 30, 35, 38). In the full-length S protein, the HR1-HR2 structure brings the fusion peptide, predicted to be near the N terminus of HR1 (4), into close proximity to the transmembrane domain, and this facilitates the fusion between viral and cellular membranes, allowing the virus to enter the cell. It is very probable that, by binding to the HR2 domain with high affinity, anti-S10 antibodies could block the interaction between HR1 and HR2 and hence prevent SARS-CoV fusion with the host cells.
As bacterially expressed proteins would be easy and cost-effective to produce on a large scale, the S10 fragment (aa 1029 to 1192) identified in this study may be an ideal vaccine candidate for SARS-CoV. In future studies, it will be critical to determine if the immunization of peptide S10 can prevent SARS-CoV replication and, more importantly, prevent disease in animal models. It is also important to delineate the precise contribution of this S region to membrane fusion. We are currently mapping monoclonal antibodies obtained from mice that have been immunized with GST-S10 proteins and determining their binding and neutralizing properties. Last, the recombinant S10 protein and anti-S10 antibodies can potentially be developed for antiviral treatments, as it has recently been shown to be possible to control SARS-CoV infection in ferrets with a neutralizing antibody directed against S (29).
ACKNOWLEDGMENTS
We thank Ng Yi Zhen, Daphne Chan, Tham Puay Yoke, Choi Yook-Wah, Hong He, Fang Zhizheng, and Li Pingping for technical assistance.
This work was supported by a grant from the Agency for Science, Technology and Research, Singapore.
C.-T.K. and A.Z. contributed equally to this article.
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