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Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, Ohio 44691-4096,1 Istanbul University, Faculty of Veterinary Medicine, Department of Virology, Avcilar 34320, Istanbul, Turkey,2 Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, Division of Viral Diseases, 1600 Clifton Road, Atlanta, Georgia 30333,3 Center for Infectious Disease Research and Vaccinology, Veterinary Science Department, South Dakota State University, Brookings, South Dakota 57007,4 Department of Medicine, Medical School, University of Massachusetts, 364 Plantation Street, Worcester, Massachusetts 016055
Received 29 May 2007/ Accepted 26 September 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we performed a comprehensive analysis of the antigenic cross-reactivity between SARS-CoV and other animal CoVs (groups 1, 2, and 3) and a group 1 HCoV (NL63). Because cross-reactivity between SARS-CoV and polyclonal antisera to antigenic group 1 animal CoVs has been previously attributed to the nucleocapsid protein (N protein) by others (44) and in our preliminary studies (H. S. Nagesha, M. G. Han, L. J. Saif, T. G. Ksiazek, L. J. Anderson, and L. Haynes, presented at the 23rd annual meeting of the American Society for Virology, McGill University, Montreal, Quebec, Canada, 2004), our focus was to delineate the nature of this cross-reactivity, to define the region of the N protein involved, and to map the boundaries of the cross-reactive antigenic sites. We further demonstrated that the observed two-way antigenic cross-reactivity is mediated only by the N protein and not the spike (S) protein, and we localized the potential cross-reactive antigenic sites in the N protein. The analyses performed allowed us to ascertain the range of group 1 CoVs (animal and human) cross-reactive with SARS-CoV and to demonstrate that the observed cross-reactivity is not universal among group 1 CoVs, because HCoV-NL63 did not cross-react with SARS-CoV in our study. Full-length SARS-CoV N-protein-based serologic assays were reported to produce false positive results with sera from healthy humans (29, 30, 51). Therefore, identification and characterization of non-cross-reactive fragments of the SARS-CoV N protein will allow development of serologic tests for screening of SARS-CoV-specific antibodies (Abs) in human patients and in animal reservoirs to exclude false positives due to the cross-reactivity with group 1 human or animal CoVs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hyperimmune antisera against animal and avian CoVs were produced in gnotobiotic pigs and guinea pigs and used in the present study. The anti-TGEV-Miller (strain M6) serum (M2), anti-TGEV-Purdue (strain P115) serum (MM973), and anti-PRCV (strain ISU1) serum (PP12) were produced in gnotobiotic pigs as combined postinfection (oral/intranasal [i.n.] exposure to live CoV) and hyperimmune (four parenteral injections of inactivated CoV) antisera. The anti-CCoV (strain UCD1), anti-FIPV (strain 79-1146), anti-BCoV (enteric strain Mebus), anti-HCoV-NL63, and anti-IBV (strain Massachusetts) sera were produced by parenteral hyperimmunization of guinea pigs with the inactivated CoVs. The last antiserum was provided by Y. M. Saif. An IBV-Massachusetts antiserum was also produced in chickens (by intratracheal inoculation with 105 50% egg infective doses of IBV-Massachusetts, followed by intravenous inoculation with at least 105 egg infective doses of IBV-Massachusetts) and kindly provided by M. Jackwood (University of Georgia). Also included was antiserum to enteric BCoV (BO12722), which was produced in a gnotobiotic calf by oral/i.n. exposure to a live, virulent BCoV-DB2 strain, followed by four subsequent intraperitoneal injections with inactivated cell culture-adapted BCoV-Mebus. Sera from mock-inoculated gnotobiotic pigs, gnotobiotic calves, chickens, and guinea pigs were used as negative controls in all assays. Mouse polyclonal SARS-CoV-Urbani antiserum was produced in BALB/c mice immunized with inactivated SARS-CoV lysate (Urbani strain) in Titermax adjuvant, followed by a boost with 18 µg of recombinant SARS-CoV nucleocapsid protein in phosphate-buffered saline (PBS), and provided by Lia M. Haynes (CDC, Atlanta, GA). Normal mouse serum (Sigma-Aldrich, St. Louis, MO) was used as a negative control serum. The hyperimmune rabbit antiserum to the SARS-CoV S protein (generated by four DNA immunizations by a gene gun with codon-optimized DNA vaccine expressing the full-length S protein of the SARS-CoV-Urbani strain) and negative rabbit sera were provided by Shan Lu (Medical School, University of Massachusetts, Worcester, MA).
A panel of human sera was used for assessment of antigenic cross-reactivity by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA). The panel included six convalescent-phase serum samples (CoV samples 3 to 8) from the WHO-confirmed SARS-patients (collected 18 [CoV samples 3 and 4] and 50 [CoV samples 5 to 8] days after disease onset) provided by Thomas Ksiazek (CDC, Atlanta, GA) and 15 serum samples (CoV samples 10 to 24) obtained from healthy donors and provided by Matthias Niedrig (Robert Koch Institute, Berlin, Germany), which qualified as SARS negative in the first PCR in the external quality assurance test. CoV sample 9 was a human serum sample obtained from an adult in the United States that we demonstrated was negative for Abs to irradiated SARS-CoV in ELISA and the SARS-CoV N and S proteins in ELISA and Western blot analysis. All serum samples were stored at –70°C until use.
MAbs. Two sets of monoclonal Abs (MAbs) were used to analyze cross-reactivity between SARS-CoV and group 1 CoVs: anti-TGEV-M6 N-protein MAbs (25H7, 14E3, 14G9) previously produced and characterized in L. J. Saif's laboratory (52) and anti-SARS-CoV-Urbani N-protein MAbs (SA 46-4 and SA 87-A1) kindly provided by Ying Fang (South Dakota State University, Brookings, SD) (7). The TGEV-M6 N MAbs recognized three distinct antigenic sites on the TGEV N protein: N1 (25H7), flanked by amino acids (aa) 1 to 120; N2 (14E3), imbedded between aa 255 and 383; and N3 (14G9), spanning from aa 1 to aa 205 (52). A MAb to SARS-CoV-Urbani spike, 341C (46), was kindly provided by Lia M. Haynes (CDC, Atlanta, GA). Negative mouse ascites SP2/0 was used as a negative control in assays with the different MAbs.
RNA extraction. The RNA was extracted from a cell culture supernatant of CoVs, including TGEV-M6, TGEV-P115, PRCV-ISU1, HCoV-NL63, and the irradiation-inactivated, cell-cultured SARS-CoV-Urbani strain, using an RNeasy mini kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA).
Cloning of the N-protein genes (full-length and fragments). Twelve oligonucleotide-containing, engineered restriction enzyme sites (EcoRI and SalI) for amplification and cloning of eight truncated and one full-length variant of the SARS-CoV N protein were designed to facilitate cloning in a prokaryotic (vector-pET23b) (Novagen, EMD Biosciences, San Diego, CA) expression system (Table 1). Using these primers, the nine fragments (including the full-length fragment) were amplified, digested with the EcoRI and SalI restriction enzymes, gel extracted, and ligated into the pET23b plasmid vector. Two more oligonucleotide pairs were designed for the full-length cloning of N proteins of group 1 CoVs, including two TGEV strains (Miller-M6 and Purdue-P115), the PRCV ISU1 strain, and the HCoV-NL63 strain (HCoV-NL63); three additional oligonucleotides were designed for HCoV-NL63 N-protein fragment cloning (Table 1). After reverse transcription-PCR, amplified fragments with these primers were digested with the EcoRI and NotI restriction enzymes, purified, and ligated into the pET23b plasmid. After transformation of the constructs into Escherichia coli strain Top Ten (Invitrogen, Carlsbad, CA), selected colonies were grown and then tested by PCR and restriction analysis. Positive clones carrying correct insertions in the pET23b plasmid vector were sequenced and used for recombinant protein expression in the E. coli host strain for expression, BL-21(DE3) (Novagen, EMD Biosciences).
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3 h). The BL-21(DE3) cells produced T7 polymerase, and the expression of the target proteins was driven by a T7 RNA polymerase promoter after addition of 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) according to the manufacturer's protocol (Novagen, EMD Biosciences). After induction, cells were incubated for 3 more hours, and then cell suspensions were centrifuged at 2,500 x g to pellet cells, washed once with cold 1x PBS, and centrifuged (2,500 x g) again. Pellets were then frozen at –20°C. Purification of recombinant proteins. Cloning in the vector of choice (pET23b) allowed expression of recombinant proteins fused with a six-His tag on the C-terminal end. HIS-Select nickel affinity gel (Sigma-Aldrich) was used for extraction of recombinant proteins, following the two protocols: one for native and one for denaturing conditions. All recombinant N proteins (rNs) expressed in E. coli BL21(DE3) formed mainly inclusion bodies and could be purified from the soluble fractions in limited quantities only. There was no difference in the antigenicities of the recombinant products purified by any of the conditions noted; thus, for all purifications after initial comparison, we used only denaturing conditions because they give rise to higher protein yields.
Denaturing conditions (batch purification method). The E. coli BL-21(DE3) cell pellets were suspended in 5 ml of 6 M guanidine hydrochloride (Gu-HCl) buffer containing 5 mM dithiothreitol (DTT) and lysed by sonication with an ultrasonic processor (Vibra cells; Sonics & Materials, Inc., Newtown, CT). The resulting lysates were centrifuged at 2,500 x g for 10 min at 4°C using an Allegra X-15R benchtop centrifuge (Beckman Coulter, Fullerton, CA). After centrifugation, the clarified supernatants were applied to 1 ml (each) of HIS-Select nickel affinity gel equilibrated with 5 volumes (5 ml/ml) of equilibration buffer (0.1 M sodium phosphate buffer [Na2HPO4-NaH2PO4], 6 M Gu-HCl, 5 mM DTT) (pH 8.0). After gentle mixing for 3 min at room temperature, the suspensions were centrifuged at 250 x g for 5 min at 4°C. The supernatants were discarded, and the gel with bound proteins was washed two times with 5 ml of wash buffer (0.1 M Na2HPO4-NaH2PO4, 6 M Gu-HCl, 5 mM DTT) (pH 6.3). Then, the six-His-tagged proteins were eluted with 2 ml of elution buffer (0.1 M Na2HPO4-NaH2PO4, 6 M Gu-HCl, 5 mM DTT) (pH 4.5).
The purity of the purified proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blot analysis. Concentrations of the purified recombinant proteins were assessed using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) based on the method of Bradford (1).
Expression of the S protein of SARS-CoV. The VRC 8304 human codon-optimized SARS-CoV S-gene expression vector was kindly provided by Gary Nabel (NIH/NIAID/VRC, Bethesda, MD) (17). Human lymphocyte 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) supplemented with 10% fetal bovine serum. For transient transfection, approximately 106 293T cells were seeded into each well of six-well plates and incubated for 24 to 48 h at 37°C. Five to ten micrograms (per well) of purified recombinant VRC 8304 was transfected using the Cellfectin reagent (Invitrogen). Briefly, 5 to 10 µg DNA and 10 µl Cellfectin were separately mixed with 100 µl of unsupplemented DMEM each. Then, DNA-containing and Cellfectin-containing solutions were combined and incubated at room temperature for 30 min. Afterward, the final volume of the mixture was adjusted to 2 ml with DMEM. The 293T cells were washed before transfection with unsupplemented DMEM, and then the DNA-Cellfectin mixture was overlaid onto the cells. At 48 h after transfection, cells were fixed using 80% acetone and used for cell culture immunofluorescence (CCIF) assays as described previously (12). To obtain larger amounts of the SARS-CoV S protein, 293T cells were grown in 175-cm2 flasks and transfected with 50 µg of VRC 8304. At 48 h postinfection, cells were harvested and used for Western blot analysis and ELISA.
Western blot analysis. The Western blot analysis was performed to verify the recombinant protein expression levels and antigenicity or to assess cross-reactivity by using the unpurified CoVs as infected cell lysates. Recombinant proteins and clarified virus- or mock-infected cell supernatants were lysed by boiling them for 5 min in 1x loading buffer (Fermentas, Hanover, MD) in the presence of 200 mM DTT. The purified or unpurified recombinant proteins (20 or 50 µg/lane, respectively) were separated by 12 to 15% SDS-PAGE and transferred to nitrocellulose membranes (trans-blot transfer medium; Bio-Rad, Hercules, CA). The membranes were subsequently blocked (overnight at 4°C) in blocking buffer (PBS, pH 7.4, and 10% nonfat dry milk [NFDM]) and incubated at room temperature for 1 h with human anti-SARS-CoV polyclonal Ab, anti-six-His tag mouse MAb (Invitrogen), or the animal CoV antisera described above. After incubation, membranes were rinsed for 20 min in PBS-0.05% Tween 20, and then the bound Abs were detected with anti-swine, anti-chicken, anti-guinea pig, anti-mouse, anti-bovine, or anti-human immunoglobulin G (IgG) conjugated with horseradish peroxidase at dilutions of 1:1,000, 1:1,000, 1:5,000, 1:1,000, 1:1,000, or 1:1,000, respectively. The immunoprecipitated bands, after rinsing for 20 min in PBS-0.05% Tween 20, were developed using the TMB membrane peroxidase substrate system (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Developed membranes were washed with distilled water and air dried.
ELISA using crude cell lysates and purified recombinant proteins. The 96-well ELISA plates (Nunc MaxiSorp high protein-binding capacity ELISA plates; Nunc, San Diego, CA) were coated (coating buffer [20 mM Na2CO3, 20 mM NaHCO3, pH 9.6]) overnight at 4°C with recombinant proteins (50 to 100 ng/well) or animal and human NL-63 CoV-infected cell lysates diluted 1:50 (obtained by two freeze-thaw cycles and then clarified by centrifugation) and purified SARS-CoV diluted 1:2,000. The wells were washed with PBS-0.05% Tween 20 and then blocked with 5% NFDM in PBS. Serially diluted sera were added and incubated for 1 h at 37°C. The plates were washed with PBS-0.05% Tween 20 and incubated for 1 h with anti-swine, anti-chicken, anti-guinea pig, anti-mouse, anti-bovine, or anti-human horseradish peroxidase-conjugated IgG at dilutions of 1:750, 1:1,000, 1:5,000, 1:500, 1:500, or 1:500, respectively. The plates were washed and developed using the TMB Microwell peroxidase substrate system (Kirkegaard & Perry Laboratories, Inc.). Then, the reactions were stopped with 0.1 M HCl, and results were read at an absorbance of 450 nm by using a SpectraMax 340PC384 ELISA reader (Molecular devices, Union City, CA).
All ELISAs described in the present study were performed under stringent conditions to avoid nonspecific reactions: all Abs were diluted using 5% NFDM and 1% Tween 20 in PBS; plates were washed five times at each step. Appropriate negative controls were included in all experiments: mock-infected host cell lines HRT-18, ST, Vero E6, CrFK, A72, LLCK12; mock-transfected 293T cells; vector pET23b backbone-transformed E. coli BL-21(DE3); allantoic fluids from uninfected specific-pathogen-free chicken embryos or homogenized baby turkey gut as a negative control for the group three CoVs; and CoV-negative gnotobiotic pig, guinea pig, rabbit, and mouse and SARS-CoV-negative human sera.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We could not confirm cross-reactivity by using only SARS convalescent-phase human sera, because the results would be compromised by the presence of Abs to other HCoVs. Therefore, SARS-CoV mouse hyperimmune antiserum was used to confirm the presence of the two-way cross-reactivity between SARS-CoV and group 1 CoVs. The results clearly demonstrated the presence of a cross-reactive antigenic site(s) for TGEV-P115 and TGEV-M6, whereas PRCV-ISU1 cross-reacted more weakly, based on the relatedness (R%) values as shown in Table 2. Although unpurified CoVs were used in this experiment at the same infectious titers, antigens still could be present in different concentrations and responsible for these variations. However, no reactivity was shown for FIPV, CCoV, HCoV-NL63, and group 2a or 3 CoVs by using the SARS-CoV mouse hyperimmune antiserum.
To demonstrate which SARS-CoV major structural proteins were responsible for the observed cross-reactivity, SARS-CoV S and N proteins and TGEV (M6 and P115), PRCV-ISU1, and HCoV-NL63 N proteins were expressed and assessed by ELISA and Western blot assays.
Cross-reactivity assessment using SARS-CoV S protein expressed in 293T cells. To test whether any cross-reactivity was mediated through SARS-CoV S protein, the VRC 8304 human codon-optimized SARS-CoV S gene expression vector was used for SARS-CoV S-protein transient expression in 293T cells. Successful expression of the S protein was confirmed by Western blot analysis, CCIF, and ELISA with MAbs or polyclonal Abs to the SARS-CoV S protein. Transfected and subsequently fixed 293T cells were assessed by CCIF. S protein-containing crude cell lysates were used in ELISA and Western blot analysis with the panel of SARS convalescent-phase (CoV samples 3 to 8) and negative human (CoV samples 9 to 24) sera. The recombinant S protein demonstrated strong immunoreactivity with all SARS convalescent-phase sera but did not react with any SARS-negative sera (Table 3). Mouse hyperimmune SARS-CoV antiserum also reacted with S protein at a high titer (1:3,200). In addition, recombinant S protein reacted only with homologous SARS-CoV antisera and no other CoVs or recombinant CoV proteins tested reacted with the SARS-CoV monoclonal or polyclonal Abs to S protein (Table 3). Thus, no cross-reactivity between SARS-CoV and other CoVs mediated by the S protein was demonstrated in any assays.
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The reactivities of all rNs were analyzed by ELISA with the panel of animal antisera to the CoVs (Table 3). The results confirmed previous findings for porcine CoV antisera: the strongest cross-reactivity was demonstrated between SARS-CoV rN and TGEV-P115 and TGEV-M6 antisera, with weaker cross-reactivity with PRCV-ISU1 antiserum. Anti-CCoV and anti-FIPV sera reacted broadly with all group 1 rNs, including HCoV-NL63 rN, but with the SARS-CoV rN, the level of cross-reactivity was below the detection threshold in ELISA, although it reacted with these sera in Western blot analysis (data not shown). No cross-reactivity was observed between the group 1 CoVs or SARS-CoV rNs and group 2a BCoV-Mebus and group 3 IBV-Massachusetts antisera. Although all group 1 CoV antisera recognized HCoV-NL63 rN, HCoV-NL63 antiserum did not cross-react with any but the homologous rN.
The ELISA results obtained using the CoV rNs and SARS convalescent-phase and negative human sera were similar to those obtained with crude CoV-infected cell lysates (Table 3), suggesting the presence of SARS-CoV N-protein specific Ab as well as cross-reactive Abs to group 1 CoV N proteins in these sera. The SARS-CoV rN discriminated positive and negative human sera. The TGEV-M6 and TGEV-P115 rNs reacted with most SARS convalescent-phase sera at low titers, but unlike for the unpurified CoVs, they did not react with the negative human sera. The level of PRCV rN reactivity with SARS convalescent-phase antisera was lower; PRCV rN reacted with low titers with half of the tested SARS convalescent-phase sera but was also undetectable with the negative human sera. The HCoV-NL63 rN reacted at high titers with both convalescent-phase and negative human sera, but with some SARS convalescent-phase sera (CoV samples 3, 4, and 6), the titers were twice as high as for negatives (Table 3). In addition, rNs from TGEV and PRCV reacted specifically with mouse-derived SARS-CoV hyperimmune antiserum at low titers (100 to 400), whereas for the SARS-CoV rN, the titer was 3,200 (Table 3).
The results from this experiment prove that cross-reactivity between SARS and animal group 1 CoVs appears to be two way. However, no cross-reactivity between SARS and porcine CoVs was shown in ELISA or Western blot analysis using SARS-CoV or TGEV-M6 anti-N MAbs. The SARS-CoV anti- N MAbs reacted only with SARS-CoV rN, and TGEV-M6 anti-N MAbs reacted with rNs of group 1 CoVs, TGEV- M6, TGEV-P115, PRCV-ISU1, and HCoV-NL63 (data not shown).
Identification of the cross-reactive region by use of recombinant fragments of SARS-CoV N protein expressed in E. coli BL-21(DE3). In addition to the full-length gene, eight truncated fragments of the N-protein gene of SARS-CoV (Fig. 1) were amplified with the designed primer pairs. These fragments were predicted (by Pepscan) to carry immunodominant antigenic sites and were shown to carry immunogenic sites in previous studies (15) or were used in commercial SARS-CoV Ab detection systems (Genesis Biotech Inc., Taiwan). One of the N-terminal short fragments, aa 120 to 208, was designed to exclude the possibility that cross-reactivity is mediated through the highly conserved motif FYYLGTGP (aa 111 to 118), which is present in the N proteins of all CoVs. The shortest C-terminal fragment (aa 360 to 412) was included in the study as the most specific region of the SARS-CoV N protein, with a unique lysine-rich amino acid stretch, KTFPPTEPKKDKKKKTDEAQ (aa 362 to 381).
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We then assessed the cross-reactivities of all SARS-CoV N-protein fragments in Western blot analysis and ELISA using the group 1 hyperimmune anti-TGEV (anti-P115 [MM973] [Fig. 2d] and anti-Miller M6 [M2]) and anti-PRCV (PP12) sera produced in gnotobiotic pigs and hyperimmune antisera to FIPV-79-1146, CCoV-UCD1, HCoV-NL63 (group 1), BCoV-Mebus (group 2), and IBV-Massachusetts (group 3), all produced in guinea pigs, and to IBV-Massachusetts produced in chickens. Strong reactivity was detected between the fragments comprising aa 120 to 208, 70 to 213 (both strongest), and 1 to 213 and the porcine CoV antisera in Western blot analysis (Fig. 2d). For the full-length N protein (1 to 422), staining was weaker but detectable (Fig. 2d) with all porcine antisera. In addition, low-level reactivity was detected between the fragment comprising aa 1 to 213 and FIPV and CCoV antisera. The ELISA results with purified SARS-CoV N-protein fragments and animal group 1 antisera were mostly consistent with Western blot analysis; however, the fragment comprising aa 1 to 213 demonstrated the strongest cross-reactivity with all animal group 1 CoV antisera and no cross-reactivity was observed for the fragments from the C-terminal part of the N protein (Table 3). No reactivity was evident for any fragments with HCoV-NL63, BCoV-Mebus, or IBV-Massachusetts antisera in either Western blot analysis or ELISA.
Assessment of the antigenicity of the putative cross-reactive region by use of recombinant fragments of HCoV-NL63 N protein expressed in E. coli BL-21(DE3). To define which HCoV-NL63 N-protein regions contribute to cross-reactivity with other group 1 CoVs and to test whether the NL63 N-protein region comprising aa 39 to 183, corresponding to the SARS-CoV N-protein cross-reactive region (based on amino acid alignment), possesses high antigenicity, three HCoV-NL63 N-protein fragments were expressed in E. coli: the N-terminal half (aa 1 to 183), the C-terminal half (aa 182 to 378), and the region comprising aa 39 to 183, which corresponds to the longer SARS-CoV cross-reactive N-protein fragment (aa 70 to 213). After successful expression was confirmed, the antigenicities of the recombinant proteins were assessed with the set of hyperimmune and SARS convalescent-phase sera in Western blot analysis and ELISA. Western blot analysis with hyperimmune guinea pig antiserum to HCoV-NL63 demonstrated the presence of a strong antigenic site in the C-terminal part of the N protein (aa 182 to 378), whereas for the N-terminal part (aa 1 to 183), a much lower antigenicity was shown and no reactivity was observed for the aa-39-to-183 fragment (Fig. 3). Hyperimmune antisera to TGEV-P115 and -M6 and TGEV-M6 anti-N MAbs 14G9 and 25H7 reacted with the N-terminal half of the HCoV-NL63 N protein but not with the aa-39-to-183 or aa-182-to-378 fragment, whereas TGEV-M6 anti-N MAb 14E3 and hyperimmune PRCV-ISU1 antiserum reacted with the aa-182-to-378 fragment (Fig. 3). Interestingly, MAbs 25H7 and 14E3 recognized distinct antigenic sites on the TGEV N protein, N1 (aa 1 to 120) and N2 (aa 255 to 383), respectively, whereas MAb 14G9 recognized a third distinct antigenic site, N3 (aa 1 to 205) (52). Human SARS convalescent-phase sera reacted strongly with the N-terminal half; weaker reactivity was observed for the C-terminal half, and no reactivity was shown for the aa-39-to-183 fragment (data not shown). The ELISA results for purified HCoV-NL63 N-protein fragments (50 to 100 ng/well) (Table 4) confirmed those of Western blot analysis by demonstrating the presence of a strong antigenic site in the C-terminal part of the N protein when tested with homologous NL63 antiserum and no reactivity for the putative cross-reactive region (aa 39 to 183) with any heterologous Abs. However, in contrast to results of Western blot analysis, antiserum to HCoV-NL63 reacted with the aa-39-to-183 fragment in ELISA at a low titer (Table 4). Although similar cross-reactivities with the N-terminal part were observed for all three antisera to porcine CoVs, the reactivities with the C-terminal half of the N protein differed dramatically between the three antisera, with the highest level detected for the PRCV-ISU1 antiserum (Table 4). In addition, no reactivity was observed between mouse hyperimmune antiserum to SARS-CoV and any of the three fragments tested in ELISA or Western blot analysis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antigenic cross-reactivity between SARS-CoV and animal group 1 CoVs was reported by several research groups (21, 44), including our preliminary studies (H. S. Nagesha et al., presented at the 23rd annual meeting of the American Society for Virology, McGill University, Montreal, Quebec, Canada, 2004), and it was attributed to the N protein (44; H. S. Nagesha et al., presented at the 23rd annual meeting of the American Society for Virology, McGill University, Montreal, Quebec, Canada, 2004). To our knowledge, no antigenic cross-reactivity has been reported between SARS-CoV and animal group 2a or 3 CoVs, which is consistent with the findings in this report based on ELISA and Western blot analysis.
Also consistent with previously published results, we confirmed that the TGEV-P115, TGEV-M6, and PRCV-ISU1 antisera (group 1) from gnotobiotic pigs cross-reacts with SARS-CoV in ELISA and Western blot analysis. Gnotobiotic pigs are free from extraneous microbes and maternal or preexisting Abs to CoVs. They are the original host species for TGEV and PRCV, reflecting the full spectrum of postinfection and posthyperimmunization-induced Abs (all pigs received oral/i.n. exposure to the CoVs and were recovered from infection prior to parenteral hyperimmunization) that may influence the Ab specificities and results. The cross-reactivity of the guinea pig antisera produced against inactivated CCoV and FIPV with SARS-CoV was lower than that for TGEV antisera, possibly because the Ab spectra induced in heterologous hosts by parenteral injection of inactivated CoVs are not equivalent to those that develop in the natural host postinfection. The SARS-CoV hyperimmune mouse antiserum allowed us to confirm the two-way cross-reactivity between SARS-CoV and group 1 CoVs, with the highest level of reactivity between this antiserum and TGEV-P115 and -M6. The broad reactivity of the group 1 HCoV-NL63 with all sera of human origin suggested the widespread presence of group 1 CoV Abs to HCoV-NL63 or the cross-reactive 229E strain in the adult sera. Because we did not observe cross-reactivity between SARS-CoV and HCoV-NL63 (although the latter reacted in one-way cross-reactivity assays with all antisera to group 1 CoVs), we concluded that the cross-reactivity observed was not a common feature for all group 1 CoVs and SARS-CoV. Of interest, NL63 is in a phylogenetically separate branch relative to the animal group 1 CoVs that we tested and is grouped with HCoV-229E and the porcine epidemic diarrhea CoV (5, 10). We were unable to test porcine epidemic diarrhea CoV due to U.S. import restrictions on animal pathogens.
The two major structural proteins and immunogens, the nucleocapsid and spike proteins, were important for assessment in regard to cross-reactivity. Both proteins were previously demonstrated to be highly immunogenic, with the S protein bearing the antigenic sites for neutralizing Abs (13, 14, 16, 57), whereas the N protein was the most abundant viral protein, with its earliest appearance during SARS-CoV infection (2, 24, 25, 45, 49). Because of the absence of cross-reactivity mediated through the SARS-CoV S protein, we focused our remaining work on investigation of the N-protein contribution to cross-reactivity. Whether other SARS-CoV structural proteins (E, M) can also contribute to cross-reactivity has not been determined.
The moderate to weak cross-reactivity between TGEV-P115, TGEV-M6, and PRCV rNs and SARS convalescent-phase sera did not provide conclusive results, due to the likely presence of Abs to group 1 CoVs in these sera, as suggested by the extensive cross-reactivities of both SARS-CoV Ab-positive and -negative human sera with the group 1 HCoV-NL63 and HCoV-NL63 rNs. However, unlike the crude CoV-infected cell lysates, the rNs did not react with negative human sera, probably because the cross-reactivities with unpurified CoVs were mediated through Abs to several antigens of group 1 CoVs and not only to the N protein. Consistent with previous findings (30, 51), SARS-CoV rN in Western blot analysis produced few positive results with several of the confirmed negative human sera; however, it allowed reliable discrimination between SARS convalescent-phase and negative human sera in ELISA. The reactivity pattern of the antisera to porcine CoVs with SARS-CoV rN was similar to those obtained with the unpurified CoVs, so we concluded that the N protein is responsible for the cross-reactivity between SARS-CoV and porcine group 1 CoVs, which is consistent with previously published results (44). However, no such conclusion could be drawn for the HCoV-NL63 rN, which reacted with both SARS convalescent-phase and negative human sera but not with mouse hyperimmune antiserum to SARS-CoV. Considering the absence of cross-reactivity mediated by the SARS-CoV S protein and the fact that immunological responses in SARS patients are predominantly directed to the N protein (2, 8, 45), we hypothesized that some cross-reactive antigenic site(s) in the SARS-CoV N protein may be responsible for the observed cross-reactivity and also that variations in their primary sequences can influence the structures of the antigenic sites (56), leading to the different levels of cross-reactivity with the group 1 animal CoVs.
Identification of the cross-reactive (with TGEV-P115, TGEV-M6, and PRCV-ISU1 antisera) fragments, aa 70 to 213, 120 to 208, and 1 to 213, confirmed the results obtained with unpurified CoVs and full-length rNs, demonstrating the strongest binding efficiency for anti-P115 and -M6 sera and lower levels of reactivity for the fragment comprising aa 1 to 213 with hyperimmune anti-CCoV and hyperimmune anti-FIPV sera. Because the observed cross-reactivity was highest for the fragment comprising aa 1 to 213 but was not greatly decreased for the shortest fragment, aa 120 to 208, we speculate that most of the cross-reactive antigenic site was imbedded within the aa-120-to-208 fragment, probably spanning beyond its borders, and that the highly conserved motif FYYLGTGP (aa 111 to 118) is not likely the source of the observed cross-reactivity, because its exclusion in the fragment comprising aa 120 to 208 did not affect cross-reactivity compared with that in the fragment comprising aa 70 to 213.
Although we did not have an opportunity to use a sufficient panel of human sera, from the results that we obtained, the SARS-CoV N-protein fragments comprising aa 1 to 213 and 360 to 412 appeared to be valuable components for a SARS-CoV Ab detection ELISA. For these fragments, 100% sensitivity and specificity were shown (using the available set of sera), and moreover, these fragments were capable of recognizing all SARS convalescent-phase sera with higher efficiency than systems based on full-length S or N protein, with the detection efficiency equal to that of the SARS-CoV based ELISA. This finding suggests that these fragments carry highly antigenic linear sites, which are the main targets for immune responses to the whole N protein and, possibly, to the intact SARS-CoV. Although the aa-360-to-412 fragment failed to react with SARS-CoV hyperimmune antiserum produced in a mouse, this does not diminish but may in fact enhance its value for SARS diagnosis in the infected human population. This failure might be related to differences in the antigen presentation or immunogenicity of these sites between humans and mice (16, 28) or the fact that the human sera represented postinfection convalescent-phase sera and the mouse hyperimmune antiserum was produced using inactivated SARS-CoV given parenterally, followed by a boost with recombinant SARS-CoV N protein. The fragment comprising aa 1 to 213 also contained immunodominant sites, and although our studies demonstrated that it was responsible for the cross-reactivity with animal group 1 CoVs, surprisingly it did not seem to contain epitopes cross-reactive with another group 1 HCoV, HCoV-NL63. This finding suggests that SARS-CoV evolution was isolated from that of HCoV-NL63 and probably was more closely related to that of the animal CoVs. However, further studies including other human and animal CoVs are required to reach a more definitive conclusion.
The observed low-level reactivity of the aa-1-to-213, aa-212-to-422, and aa-1-to-422 fragments with some human negative sera in Western blot analysis did not appear to be related to sites that shared higher sequence identity between SARS- and other HCoVs, because staining was equally weak for all three fragments and could be due to other reasons, including possible N-protein sequence identity with some human tissues (51), cross-reactivity with some undetected human pathogen, or a nonspecific affinity of IgG to the recombinant nonglycosylated N protein (29, 30).
Alignment of the amino acid sequences of the three porcine CoV and SARS-CoV N proteins did not reveal major differences potentially responsible for the observed variations in cross-reactivity levels for the animal group 1 CoVs (TGEV-P115, TGEV-M6, PRCV), because the N-protein sequence is highly conserved within groups and all three porcine group 1 CoV N proteins possessed 97% amino acid identities between one another, with infrequent point mutations (58). Only 1 out of 12 polymorphic positions for the TGEV-M6, TGEV-P115, and PRCV-ISU1 N proteins appeared to be critical: a valine (V) at aa position 120 in the PRCV-ISU1 N protein (within the cross-reactive region) might have diminished its cross-reactivity with SARS-CoV, because both TGEV (M6 and p115) and SARS-CoV contain alanine (A) at this position (58). Considering that the cross-reactivity observed may not be a direct consequence of the amino acid sequence identity, one common feature of N proteins from TGEV, PRCV, and SARS-CoV should be noted: the presence of a strong immunodominant site in the N-terminal (cross-reactive) region. This was previously reported for the SARS-CoV (15), and we found similar results for TGEV and PRCV (unpublished data). The TGEV and SARS-CoV N proteins were shown to be RNA chaperons with long disordered regions (60). Similar, very short antigenic sites (as opposed to conformational sites that would have been lost during protein purification under the denaturing conditions that we used) are probably exposed during natural infection in the host species. This, together with the overall low identity of the primary sequences of SARS-CoV and porcine CoVs, suggests that one point mutation within a cross-reactive region can be critical for the cross-reactive site structure and responsible for the observed variations in the levels of cross-reactivity with porcine group 1 CoVs. Interestingly, the most antigenic site of the HCoV-NL63 rN, as reflected by the hyperimmune antiserum, was located in the C terminus, whereas cross-reactivity with group 1 CoVs was mediated through at least two antigenic sites in the N (presumably aa 1 to 39) and C (aa 182 to 378) termini of the HCoV-NL63 N protein, whereas the putative cross-reactive (aa 39 to 183) N-protein fragment of HCoV-NL63 failed to react with any heterologous Abs. Therefore, this fragment of the HCoV-NL63 N protein differed in its antigenicity and antigenic cross-reactivity from the SARS-CoV and animal group 1 CoVs. This could explain the absence of cross-reactivity with SARS-CoV, but as noted for animal group 1 CoVs, the determinants of antigenicity could not be identified at the primary amino acid sequence level.
In conclusion, we confirmed the two-way antigenic cross-reactivity between SARS-CoV and porcine group 1 CoVs (TGEVs [M6 and P115] and PRCV-ISU1) and demonstrated that it was attributable to the SARS-CoV and group 1 CoV N proteins and not the S protein. We identified the cross-reactive region and one point mutation within this region likely responsible for the observed variations in the levels of cross-reactivity with porcine group 1 CoVs. In addition, our findings suggest that the fragments comprising aa 1 to 213 and 360 to 412 of the SARS-CoV N protein when used together can be valuable discriminatory diagnostic reagents, with the latter reflecting only SARS-CoV specificity. Use of such paired reagents could overcome the problems of nonspecificity demonstrated for the full-length N protein for testing both human and animal sera.
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ACKNOWLEDGMENTS |
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This work was supported by grant R21 AI062763 from the NIAID, NIH. Salaries and research support were provided by state and federal funds to the Ohio Agricultural Research and Development Center, The Ohio State University.
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FOOTNOTES |
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Published ahead of print on 3 October 2007.
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