Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers

Materials.

All reagents used in this study were purchased from Sigma (St. Louis, Mo.), unless otherwise stated. All cell lines were purchased from the American Type Culture Collection (Manassas, Va.) and were cultured at 37°C in 5% CO₂ in Dulbecco's modified Eagle medium containing 1 g of glucose/liter, 2 mM l-glutamine, 1.5 g of sodium bicarbonate/liter, 0.1 mM nonessential amino acids, 0.1 mg of streptomycin/ml, 100 U of penicillin, and 5% fetal bovine serum (HyClone, South Logan, Utah).

Construction of plasmids.

For transient expression in mammalian cells, the vectors used were pXJ40HA, for tagging proteins at the N terminus with one hemagglutinin (HA) epitope (8), and pXJ40-3′HA, for tagging proteins with three HA epitopes at the C terminus (T. Leung, Institute of Molecular and Cell Biology, Singapore, Republic of Singapore, personal communication). For the expression of glutathione S-transferase (GST) fusion proteins in bacteria, genes were cloned in frame with GST in pGEX-4T-1 (Amersham Pharmacia Biotech, Uppsala, Sweden). For the stable transfection of S in CHO cells, a full-length S construct tagged at the C-terminal end with green fluorescent protein (GFP) was cloned into the pMMTC vector (14). All of the constructs used for this study are listed in Table 1.

SARS-CoV 2003VA2774, an isolate from a SARS patient in Singapore, was used for this study. For the cloning of various genes, RNAs were extracted by use of a Qiagen viral RNA kit (Valencia, Calif.) from a SARS-CoV-infected Vero E6 cell culture supernatant harvested when the cultures showed at least 75% cytopathic effects. Reverse transcription was performed with Superscript II RNase H⁻ reverse transcriptase (Gibco BRL, Gaithersburg, Md.) and an oligo(dT) primer. PCRs were performed with either HotStar polymerase (Qiagen), Titanium Taq DNA polymerase (Clontech Laboratories Inc., Palo Alto, Calif.), or High Fidelity Taq polymerase (Roche Molecular Biochemicals, Indianapolis, Ind.). In some cases, overlapping cDNAs provided by the Genome Institute of Singapore (isolate SIN2774; accession no. AY283798 [12]) were used as templates instead.

All of the primers used for this study were synthesized by Genset Singapore Biotech (Singapore). The sequences of all constructs used in this study were confirmed by DNA sequencing performed at the core facility at the Institute of Molecular and Cell Biology by the dideoxy method with a Taq DyeDeoxy terminator cycle sequencing kit and an automated DNA sequencer (model 373) from PE Applied Biosystems (Foster City, Calif.).

Transient transfection of mammalian cells.

Transient transfection experiments were performed with Effectene transfection reagent (Qiagen) according to the manufacturer's protocol. Typically, ∼10⁶ COS-7 cells were plated on a 6-cm-diameter dish and allowed to attach for at least 4 h. One to 2 μg of DNA was used per plate, and the cells were left for at least 14 h before the cells were washed with phosphate-buffered saline (PBS), lysed directly in Laemmli's sodium dodecyl sulfate (SDS) buffer, and used for Western blot analysis.

Expression of GST fusion proteins.

Exponentially growing cultures (optical density at 600 nm of ∼0.7) of Escherichia coli (BL21/DE3) cells harboring the pGEX-4T-1 expression constructs were induced to synthesize fusion proteins by the addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), after which the cells were allowed to grow for another 4 h at 37°C or 12 h at 30°C. Cells were harvested, resuspended in PBS containing 0.5% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride, and then sonicated with an ultrasonic processor (Misonix Inc., Farmingdale, N.Y.). GST fusion proteins were then purified from the lysate by use of glutathione (GSH)-Sepharose beads (Amersham Pharmacia Biotech). Proteins were eluted from the beads with 10 mM reduced GSH in 50 mM Tris-HCl, pH 9.2, and 0.1% SDS, and protein concentrations were determined by use of a Coomassie Plus assay kit (Pierce, Rockford, Ill.). Proteins were also separated in SDS-polyacrylamide gels and stained with 0.25% Coomassie brilliant blue R-250 (Bio-Rad, Hercules, Calif.) in 45% methanol and 10% acetic acid.

Western blot analysis.

For Western blot analysis, approximately 10⁵ transfected COS-7 cells or 50 ng of GST fusion proteins was separated in SDS-polyacrylamide gels and transferred to nitrocellulose Hybond C membranes (Amersham Pharmacia Biotech). The membranes were blocked with 5% nonfat dry milk. For the detection of HA-tagged or GST fusion proteins, the membranes were incubated with either an anti-HA polyclonal or anti-GST monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) overnight at 4°C and washed extensively with PBST (PBS containing 0.05% Tween 20), followed by incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce) for 1 h at room temperature. Membranes were then washed extensively with PBST, and the detection of signals by an enhanced chemiluminescence method (Pierce) was performed. For patient sera, each sample was first treated with 0.5% Triton X-100 and 0.1 mg of RNase (Sigma)/ml and then diluted 1:150 to 1:500 with PBST containing 1% nonfat dry milk. After incubation for 1 to 3 h at room temperature or overnight at 4°C, the membranes were incubated with an anti-human HRP-conjugated immunoglobulin G (IgG) (Santa Cruz Biochemicals), IgA, or IgM (Zymed Laboratories Inc., San Francisco, Calif.) antibody for 1 h at room temperature, followed by detection as described above. All secondary antibodies were used at a 1:2,000 dilution. Due to the limited amount of patient sera from the seven pairs of sera, Western blots of a mixture of three GST fusion proteins, N, U274, and U122, and GST were run in a single-slot SDS-10% polyacrylamide gel electrophoresis (PAGE) gel and transferred onto nitrocellulose membranes. The membranes were cut into strips of about 0.8 mm wide, and each strip was probed with 300 to 400 μl of diluted sera. For the late time point sera, the strips were incubated with the diluted sera for 1.5 h at room temperature. For the early time point sera, the strips had to be incubated overnight with the sera, at room temperature, to detect any signal. The secondary antibodies were incubated for 1 h at room temperature. This method was also used when large numbers of samples were being screened.

Expression of S-GFP fusion protein in CHO cells and immunofluorescence analysis to determine anti-S immunoreactivity in patient sera.

CHO cells were transfected with pMMTC-S-GFP by use of DMRIE-C reagent (Gibco BRL) according to the manufacturer's protocol. Transfected cells were selected in Geneticin (Gibco BRL) for >1 week. Cells were then analyzed under a Zeiss microscope (Carl Zeiss Vision GmbH, Hallbergmoss, Germany), and the clone with the strongest expression signal was picked and grown in medium containing 100 μM ZnSO₄. Zn²⁺ ions increase the expression of genes by the pMMTC vector (14).

Cells were dislodged from the plates with 0.04% EDTA, seeded onto black Teflon Menzel diagnostic slides (Merck), and blown dry, followed by fixing in acetone at −20°C for 1 h. Then the cells were incubated with sera at dilutions of 1:20, 1:40, 1:80, and 1:160 (in PBS) for 1.5 h at 37°C, followed by incubation with a fluorescein isothiocyanate (FITC)- or rhodamine (Rh)-conjugated anti-human IgG (Sigma) at a 1:20 dilution for 1.5 h at 37°C. When Rh-conjugated anti-human IgG was used, it was diluted in PBS, and when FITC-conjugated anti-human IgG, IgM (Sigma), or IgA (Dako A/S, Glostrup, Denmark) was used, it was diluted in 0.05% Evans Blue solution (Fluka, Buchs, Switerland), which blocks GFP fluorescence from transfected cells. Slides were then viewed under a Zeiss microscope (Carl Zeiss Vision GmbH) and scored as follows: +++, very strong staining; ++, moderate staining; and +, weak staining.

Collection of sera from SARS-CoV-infected patients.

The first six serum samples (from patients 1 to 6), except for that from patient 3, were obtained by plasmapheresis. Plasmapheresis was performed on patients who (i) had previous documented SARS-CoV infection according to World Health Organization (WHO) criteria, (ii) had been in the convalescent phase for at least 6 weeks, and (iii) were symptom-free and willing to participate in the study. Blood was taken from patient 3, and the serum was separated out for the experiments. All participants have given written consent, and approval from the Tan Tock Seng Hospital Ethics Committee has been granted.

The second set of sera consisted of seven pairs of samples that were collected from patients admitted to Tan Tock Seng Hospital or Singapore General Hospital. These patients were admitted upon the onset of illness, as defined by WHO criteria, and the dates of admission and dates of collection of blood are shown in Table 2. The first samples (set A) were collected 2 to 11 days after the onset of illness, and the second set of samples (set B) were collected 16 to 54 days after the onset of illness.

The last set of sera was obtained from 61 probable SARS patients who were discharged from Tan Tock Seng Hospital. For some of these patients, sera were taken upon discharge (3 to 4 weeks after the onset of illness), while most of the patients were recruited >3 weeks after discharge (>6 weeks after the onset of illness) (see Table 3 for details). For one patient (patient P3L), a serum sample was taken 111 days (3 1/2 months) after the onset of illness. Samples from two other patients (P7 and P8) were obtained by plasmapheresis.

A control serum was purchased from Sigma, and 99 serum samples were obtained from healthy donors who have given informed consent. These healthy donors (i) did not have a diagnosis of SARS, suspect SARS, or have contact with a person who was served a home quarantine order for SARS; (ii) did not have a fever, symptoms of influenza, runny nose, or sore throat within the last week at the time of donation; (iii) had not been on immunosuppressants; (iv) had no significant medical illnesses; and (v) had no history of travel to SARS-affected areas since November 2002.

Detection of IgG antibodies against viral proteins encoded by the SARS-CoV genome in the sera of probable SARS patients.

In order to assess which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for the development of a serological assay to detect SARS-CoV infection, we cloned three of the four structural proteins (E, M, and N) into expression vectors. Due to the large size (∼4.7 kbp) and heavy glycosylation of the spike (S) protein, it was analyzed separately by an immunofluorescence method (see below). In addition, two of the SARS-CoV unique proteins (U274 and U122) were also included in this study, and they will be referred to hereafter as UX, where “X” stands for the number of predicted amino acids of the proteins. In this study, we used only the C-terminal hydrophilic region of U274, as it has three potential transmembrane domains at its N-terminal end and these hydrophobic regions may affect the solubility of a recombinant U274 protein. The positions of the structural and unique proteins and their corresponding ORF numbers, as designated by Marra and coworkers (9), are shown in Fig. 1a.

This panel of HA-tagged proteins was expressed in COS-7 cells by transient transfection, and total protein lysates were analyzed by Western blot analysis with sera from three convalescent patients (Fig. 1b, patients 1 to 3) to determine if these sera had any antibodies against the expressed proteins. Plasmapheresis was performed on patients 1 and 2, and the plasmapheresis products were used in Western blot analysis, while for patient 3, serum was used for the analysis. All three patients had antibodies against the N (amino acids [aa] 69 to 422) protein, but not against the other structural proteins, M and E. Interestingly, the C-terminal hydrophilic region of U274 (aa 134 to 274) was also detected by the sera of patients 1 and 3 but not by the serum of patient 2 (Fig. 1b). A control serum did not detect N, U274, or any other proteins. U122 was also not detected by any of the three sera, suggesting that it may not be expressed, it is not a structural protein, or it is not sufficiently antigenic.

As bacterially expressed proteins are easier and cheaper to produce on a large scale, we next expressed N and U274 as GST fusion proteins and tested them for reactivity to patient sera. Coomassie staining showed that GST-N (aa 120 to 422) and GST-U274 (aa 134 to 274) were of >90% purity after a one-step purification with GSH-Sepharose beads (Fig. 2a). The GST-N (aa 120 to 422) protein used here lacked the N-terminal part of the N protein, which contains a highly conserved motif (FYYLGTGP; aa 111 to 118 of SARS-CoV N) found in all coronaviruses (11), so there would be less chance of cross-reactivity with antibodies against other coronaviruses. From a 400-ml bacterial culture, ∼10 mg of GST-N and ∼2 mg of GST-U274 could be obtained, respectively. GST-U122 (aa 16 to 111) (lacking the signal peptide at the N terminus and the hydrophobic C terminus) and GST were also expressed and used as controls. The GST fusion proteins were probed with the same three patient sera and one control serum, as for the mammalian expressed proteins (Fig. 1b). Consistent with the proteins expressed in COS-7 cells, GST-N was clearly detected by all three patient sera and GST-U274 was detected only by the sera from patients 1 and 3 (Fig. 2b). Neither GST-U122 nor GST showed any background with all three patient sera, and there was no nonspecific binding of the control serum to any of the proteins (Fig. 2b). Sera from another three convalescent patients (patients 4 to 6) were also tested, and they were reactive specifically towards both GST-N and GST-U274, but not to GST-U122 or GST (Fig. 2b).

Profile of IgG, IgM, and IgA antibodies against N and U274 proteins in seven pairs of sera obtained at two time points of infection.

A set of paired sera from seven patients were also examined for reactivity against GST-N and GST-U274. These were obtained at two time points of infection, one at 2 to 11 days after the onset of illness (set A) and another at 16 to 54 days after the onset of illness (set B) (Table 2). For all seven cases, the second time point samples were taken at least 8 days after the first time point samples. As shown in Fig. 3a, anti-IgG antibodies against GST-N protein were present in all seven patients' sera at the later time point (set B), but not at the early time points (set A). For both sets of sera, the membranes were incubated with the diluted sera (1:150) for 1.5 h at room temperature, followed by incubation with the secondary antibody (HRP-conjugated anti-human IgG) for 1 h at room temperature. In addition, all seven of the late time point sera also contained antibodies against GST-U274, albeit at a lower level than that for GST-N (Fig. 3a, set B).

We repeated the experiment for the early time point sera (set A) but incubated the blots with diluted sera overnight at room temperature instead of for 1.5 h and used three different secondary antibodies, anti-IgG, anti-IgM, and anti-IgA, and the results for each of them are shown in Fig. 3b. For IgG, only samples 9A and 11A showed some reactivity to GST-N. For IgA, though, all seven samples showed reactivity to GST-N, and the signals were particularly strong for samples 9A and 11A. For IgM, all seven samples showed a low level of reactivity to GST-N. As the early time point sample for each patient was collected a different number of days after the onset of illness, our results suggest that by as early as 2 days after the onset of illness (patient D2, sample 2A), IgM and IgA antibodies against the N protein were present. By 9 days after the onset of illness, very high levels of IgA antibodies against N were present and IgM and IgG antibodies were also detectable (Fig. 3b, samples 9A and 11A). As for GST-U274, some reactivities were observed for IgA (samples 5A, 8A, 9A, and 11A) and for IgM (sample 11A) from 5 to 11 days after the onset of illness. Interestingly, quite a strong signal for GST-U274 was observed for sample 11A (11 days after the onset of illness) when IgM was used, and for the corresponding early sample, 11B, the signal for GST-U274 was also strong (Fig. 3).

Determination of the sensitivity and specificity of immunoreactivity (IgG) against N and U274 proteins.

In order to determine the sensitivity and specificity of the immunoreactivity against N and U274, we obtained another 61 samples from probable SARS patients who were discharged from hospitals and tested them for the presence of IgG antibodies against N and U274 by Western blot analysis. As shown in Table 3, the samples were taken from 31 to 111 days after the onset of illness. Plasmapheresis products from 2 of the patients (P7 and P8) and sera from the other 59 patients were used. All of the samples showed strong immunoreactivity against the N protein (100%), and 44 of the 61 samples (72%) were positive for U274 (Table 3). In general, the signals observed for the U274 protein were much lower than those for the N protein. The exceptions were samples from patients 749 and 859, for which the signals for the U274 and N proteins were equally strong. Another 99 samples from healthy donors were also tested, and none of them showed any immunoreactivity to N or U274. In addition, samples from two patients (patients 868 and 873) who had close contact with probable SARS patients but never developed any of the clinical symptoms also did not show any immunoreactivity to N or U274 (Table 3). Therefore, the immunoreactivity against the N protein is highly sensitive (found in 100% of a total of 81 samples from probable SARS patients) and specific.

Detection of IgG antibodies against SARS-CoV S protein in convalescent-phase sera by immunofluorescence.

Due to the large size and heavy glycosylation of the S protein, it is advantageous to express this protein in mammalian cells instead of bacteria, as it is then possible to detect antibodies that may be dependent on conformation and/or glycosylation of the S protein. CHO cells were stably transfected with a full-length S construct tagged at the C-terminal end with GFP. After selection with antibiotics, a pool of clones expressing significant levels of S protein, as indicated by GFP fluorescence, were obtained (Fig. 4). These CHO cells were used for an immunofluorescence staining method to determine if there were IgG antibodies against the SARS-CoV S protein in convalescent-phase sera. Briefly, the cells were fixed with acetone and then incubated with diluted patient sera, followed by incubation with an Rh-conjugated anti-human IgG antibody. As shown in Fig. 4, sera from patients 1 to 6 all contained IgG antibodies against S, as detected by immunofluorescence, and no signal was observed when a control serum was used.

All remaining samples were tested similarly, except that the Rh-conjugated anti-human IgG antibody was replaced with a FITC-conjugated one in the presence of a high concentration of Evans Blue solution (0.05%). This is because of the greater ease of judging cells stained with FITC than those stained with Rh, and this concentration of Evans Blue solution was sufficient to block out all of the GFP fluorescence in the transfected cells (data not shown). As shown in Table 3, 100% of the convalescent-phase sera (74 samples) showed immunoreactivity against S at a dilution of 1:40, and 100 samples from healthy donors did not show any signal at the same dilution. The lowest dilution tested was 1:20, but a few of the samples from healthy donors showed weak signals at this concentration; therefore, the results from the 1:40 dilution were used for comparison. At higher dilutions (1:80 and 1:160), weaker signals were observed for most of the patient sera, as would be expected. In particular, for samples 432, 633, 784, 840, 878, 893, 3B, 5B, and 8B, for which very strong signals (+++) were observed at a 1:40 dilution, the signals decreased gradually at higher dilutions, i.e., moderate signals (++) were observed at 1:80 and weak signals (+) were observed at 1:160.

The seven early time point samples (Table 2, set A) were also analyzed in the same manner, but with IgG, IgM, and IgA secondary antibodies separately, but none of them showed any reactivity (at a 1:40 dilution) (data not shown).