Abstract
Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterizedfor reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipi-tation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and ninewere chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and Eproteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490–510 inthe S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270–350. The mAbs charac-terized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease
1. IntroductionCoronaviruses (CoVs) are large, enveloped, positive-stranded RNA viruses that cause a variety of illnesses inhumans and animals (Lai, 1990; Lavi et al., 1999; Perlman,1998; Snijder and Horzinek, 1993; Snijder et al., 1993; Zhouet al., 2004). Most human coronaviruses (HCoVs) fall in to one of two serotypes, OC43-like and 229E-like; however, the global outbreak of severe acute respiratory syndrome (SARS) was quickly linked to infection with a novel CoV, SARS-CoV(Ksiazek et al., 2003; Peiris et al., 2003), and HCoV-NL63,has been recognized recently as a human pathogen (van derHoek et al., 2004). The CoV genome encodes numerous non-structural proteins and four or five structural proteins includ-ing the spike (S), nucleocapsid (N), membrane (M), smallenvelope (E), and (in some) strains a hemagglutinin-esterase(HE) protein (Lai, 1990). SARS-CoV has four structural pro-teins, the S, N, M, and E proteins that have various functions.The S protein forms spikes on the virion surface and is crucialfor viral attachment and entry into the host cell. It also inducesprotective immunity, and is associated with host range, tissuetropism and virulence (Sanchez et al., 1999). The N proteinforms the nucleocapsid; the M protein interacts with the nu-cleocapsid and forms the internal viral core; and the E proteinis associated with the viral envelope. SARS-CoV is geneti-cally distinct from previously described coronaviruses, whichhave been placed into three antigenic groups: I, II, and III.Human coronaviruses, i.e., 229E-like and OC43-like, belongto groups I and II, respectively, and are recognized as the sec-ond most common cause of upper respiratory disease, but areassociated infrequently with serious lower respiratory tractdisease (El-Sahly et al., 2000; Hendley et al., 1972,Makelaet al., 1998; Falsey et al., 2002). However, SARS-CoV isusually associated with serious lower respiratory tract dis-ease, having a fatality rate as ranging between 10% and15% that may be as high as 50% in patients >60 years ofage (Drosten et al., 2003; Enserink, 2003; Holmes, 2003;Ksiazek et al., 2003; Poutanen et al., 2003; Rota et al., 2003).Although the global spread of SARS-CoV was stopped inJune 2003, six instances of laboratory-acquired infectionshave been confirmed and a cluster of sporadic cases havebeen detected in Guangdong Province, China, between De-cember 2003 and April 2004 (Liang et al., 2004), demonstrat-ing the potential for SARS to re-emerge and possibly becomepandemic.Anticipating the need for improved immunologicalreagents to aid identification and characterization of SARS-CoV, monoclonal antibodies (mAbs) to SARS-CoV proteinswere produced. This report describes nine such mAbs thatinclude antibodies reactive against each of the four structuralproteins, including two S protein reactive antibodies that neu-tralize SARS-CoV.2. Materials and methods2.1. BiosafetyAll work with live SARS-CoV was done in biosafety level3 (BSL-3) containment laboratories at the Centers for DiseaseControl and Prevention, Atlanta, Georgia. SARS-CoV wasinactivated by60Co gamma irradiation at 2×106rad prior toits use as an immunogen or as an antigen in an ELISA. Withinthe limits of detecting viable virus, 2×106rads gamma irra-diation was sufficient to inactivate all infectivity.2.2. Virus preparationVero E6 cells were maintained in Dulbecco’s minimal es-sential media (DMEM, Invitrogen Corp., Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine sera (FBS,Hyclone, Logan, UT) and 2 mMl-glutamine (Invitrogen).The Urbani strain of SARS-CoV was plaque-purified, grownto stock titers in Vero E6 cells, purified by polyethylene gly-col (PEG) precipitation as described previously (Kiley et al.,1980), and frozen at−70◦C until use. Viral antigen usedfor ELISA was prepared by detergent extraction of SARS-CoV-infected Vero E6 cells and subsequent gamma irradia-tion (Ksiazek et al., 2003).2.3. B cell hybridoma productionImmunizations were performed in accordance with theguidelines of the Institutional Animal Care and Use Commit-tee. Female, 4–6-week-old, specific pathogen-free BALB/cmice (Jackson Laboratories, Bar Harbor, ME) were intraperi-toneally (i.p.) administered 200l of PEG-purified SARS-CoV inactivated by gamma-irradiation and diluted in PBS,followed by two similar immunizations 3 and 6 weekslater. Mice were euthanized by exsanguination under Avertin(2,2,2-tribromoethanol) anesthesia 3 days after the last im-munization, and their spleens were removed.Splenocytes were fused with non-secretor SP2/0 myelomacells at a 2:1 ratio using 50% polyethylene glycol 1000 (PEG1000, Sigma Chemical Company, St. Louis, MO) and cul-tured in 24-well plates as described previously (Reimer et al.,1984). B cell hybridomas were selected using DMEM supple-mented with 10% heat-inactivated FBS, 2 mMl-glutamine,and 1×sodium hypoxanthine, aminopterin, and thymidine(HAT; Invitrogen), and culture fluids were screened for reac-tivity against a SARS-CoV infected Vero E6 cell lysate andagainst a similarly prepared uninfected Vero E6 cell lysateby ELISA. The mean optical density (OD) of the mAbs re-active to SARS-CoV infected Vero E6 cell lysate (positivecontrol, P) was divided by the mean OD of mAbs reactive toa similarly prepared uninfected Vero E6 cell lysate (negativecontrol, N) and were termed P/N ratios. B cell hybridomacells from wells that gave positive-to-negative (P/N) ratios≥3 were cloned by limiting dilution. Hybridomas cell lineswere grown in DMEM supplemented with 10% FBS.Monoclonal immunoglobulin class and isotype were de-termined by use of the IsoStrip mouse mAb isotyping kit(Roche Diagnostics Corporation, Indianapolis, IN) and con-firmed for isotype by ELISA (Amersham Biosciences, Pis-cataway, NJ) as described by the manufacturer.2.4. Recombinant proteins2.4.1. Recombinant SARS-CoV N proteinEscherichia coliBL21 cells (Invitrogen) were transformedwith an inducible pET vector expressing 6×histidine-taggedSARS-CoV N protein (Invitrogen), and expressed 6×histidine-tagged SARS-CoV N protein was purified bymetal-chelate chromatography (ProBond resin; Invitrogen)following the manufacturer’s recommended protocols. Thepurified 6×histidine-tagged SARS-CoV N protein was-eluted in lysis buffer (100 mM sodium phosphate monobasic,10 mM Tris–HCl, 300 mM NaCl in PBS containing EDTA-free proteinase inhibitor (pH 8.0) adjusted to pH 4.5. The elu-ate was immediately adjusted to pH 7.5 and dialyzed againstPBS. The purified N protein was tested by ELISA for reac-tivity to hyperimmune mouse sera and anti-SARS N mAb.2.4.2. Synthetic SARS S protein fragmentsThe full-length soluble portion of the SARS-CoV Sprotein (amino acids 1–1190; S1190) and S protein frag-ments containing amino acids 1–269, 1–350, 1–490, 1–510and 270–510 (S269,S350,S490,S510,S270–510) were syn-thesized, expressed, and purified as described previously(Babcock et al., 2004). Protein fragments were analyzed byCoomassie staining and by Western Blot using human SARS-convalescent serum or mouse anti-synthetic S protein for de-tection. The convalescent serum recognized all the S pro-tein fragments with the exception of S269, while the anti-synthetic S protein recognized all the S protein fragments.2.5. Construction of replicons expressing SARS-CoV S,M, N, and E proteinsRNA was extracted from a preparation of the Urbani strainof SARS-CoV in Trizol LS reagent (Invitrogen) followingthe manufacturer’s protocol. Reverse transcription reactionsfor generation of SARS-CoV cDNA were performed witha Super Script III kit (Invitrogen) using random hexamers.The SARS-CoV cDNA was PCR-amplified with the gene-specific primer pairs using Platinum Taq Hifi (Invitrogen).Following 18 cycles of amplification, a sample of each reac-tion was analyzed by gel electrophoresis, and fragments ofthe appropriate size were noted for each of the four genes(data not shown). The gene PCR products were sequencedand the expected sequences were confirmed. The SARS-CoVM, N, and E gene PCR products were cloned directly into thevenezuelan equine encephalitis (VEE) replicon vector usingEcoRV andAscI restriction sites. The SARS-CoV S genePCR product was directly cloned into the VEE replicon vec-tor usingAscI andPmeI restriction sites. RNA was in vitrotranscribed from each SARS gene replicon DNA using a Ri-boMax T7 RNA transcription kit (Promega) following themanufacturer’s protocol.2.6. Venezuelan equine encephalitis (VEE) repliconsexpressing SARS-CoV S, M, N, and E proteinsVero cells were electroporated with replicon RNA ex-pressing either SARS M, N, S or E genes. After electropora-tion, the cells were incubated in media containing OptiPROmedia (Invitrogen) for 16 h at 37◦C. Electroporated cellswere mixed at a 2.3:1 ratio with non-electroporated cells and50l of the cell mixture was placed on each spot of a 12-wellspot slide (VWR International, Buffalo Grove, IL). The slideswere incubated at 37◦C for 3.5 h, washed with PBS, and thenfixed with acetone and methanol (1:1) for 5 min at room temperature. The fixed monolayers were analyzed in an indirectimmunofluorescence assay (IFA), using SARS convalescenthuman plasma as the primary antibody and Alexa Fluor 488chicken anti-human IgG (H + L) (Molecular Probes, Eugene,OR) as the secondary antibody. Cells electroporated with theVEE replicon RNAs containing the SARS-CoV S, M, N, andE genes were intensely fluorescent in these assays (data notshown).2.7. IFAIFA was performed on 2% paraformaldehyde-fixed VeroE6 cells infected with 1000 TCID50SARS-CoV for 27 h,mock-infected Vero E6 or Vero E6 cell lines expressingSARS-CoV S, N, M or E as described previously (Harcourtet al., 2004).2.8. ELISAAn indirect ELISA using detergent-extracted SARS-CoVinactivated by gamma-irradiation or similarly prepared VeroE6 cell lysate was used to determine the mAb reactivity asdescribed previously (Ksiazek et al., 2003). A similar ELISAprotocol was used to detect mAb reactivity to recombinantSARS-CoV N protein, untransformedE .coliBL21 cell lysatecontrol, S1190protein, or angiotensin-converting enzyme 2antigen control.2.9. RadioimmunoprecipitationSARS-CoV-infected Vero E6 cells or mock-infectedVero E6 cells (2.5×105) were radiolabeled with [35S]-methionine/cysteine (0.1 mCi/ml; ICN, Irvine, California)and reacted with individual mAbs to determine antigen speci-ficity as described previously (Benaroch et al., 1995). Briefly,radiolabelled cells were collected by centrifugation and lysedin lysis buffer (0.1% SDS; 1.0% TritionX-100; 1% NaDOC,150 mM NaCl, 10 mM Tris–HCl (pH 7.2) and 5 mM EDTA)containing 1 mM PMSF and 100 mM Pefablock (BoehringerMannheim, Mannheim, Germany). Antigen-antibody com-plexes were precipitated using Protein G sepharose beads(Amersham Biosciences, Uppsala, Sweden), and the boundradiolabeled immunoprecipitate was analyzed by 12.5%SDS-polyacrylamide gel electrophoresis (SDS-PAGE).2.10. MAb epitope mappingImmunoprecipitation and Western Blot analysis were em-ployed to determine the epitopes recognized by the anti-Sprotein mAbs. HEK-293 cells were transiently transfectedwith DNA encoding various S protein fragments (S269,S510,S490,S350,S270–510,S1190), and culture supernatants wereharvested 48 h post-transfection. Equal amounts of S protein-containing supernatants were incubated with respective anti-S protein mAbs and protein G-Sepharose for 2 h at roomtemperature while shaking. The beads were washed in PBS and protein was eluted in Laemmli sample buffer by boil-ing the sample for 5 min. Samples were resolved by 10%SDS-PAGE and transferred to an Immobilon P membrane(Millipore, Billerica, MA). Membranes were washed, using1% non-fat dried milk in PBS–Tween, and incubated withanti-His6 mAb (Invitrogen). Blots were washed twice in 1%non-fat dried milk in PBS–0.05% Tween 20, incubated withhorseradish peroxidase-conjugated (HRP) anti-mouse IgG(Jackson Laboratories, Bar Harbor, ME) for 45 min at roomtemperature, and washed, and bands were detected by use ofan enhanced chemiluminescence reagent (Amersham) andexposed to X-Omat-AR film for various times. For Westernblotting, 1 ml of each S protein fragment supernatant was in-cubated with Ni-NTA agarose (Invitrogen) for 2 h at roomtemperature. Resin was washed with PBS, mixed with re-ducing Laemmli buffer, and separated by 10% SDS-PAGE.The proteins were transferred to Immobilon P (Millipore);the membranes were blocked using 5% non-fat dried milk inPBS–Tween overnight, and incubated for 1 h with respectiveanti-S protein mAbs. The blots were washed and incubatedwith HRP-conjugated anti-mouse IgG. After being washed,the bands were detected as described above (Babcock et al.,2004).2.11. Virus microneutralization assayThe mAbs were tested for their ability to neutralize SARS-CoV infection of Vero E6 cells by microneutralization assayas described (Sui et al., 2004). The microneutralization titer oftest antibody was the reciprocal of the highest dilution of testantibody that showed inhibition in all triplicate wells. Con-trols were included for each microneutralization assay per-formed and included back titration, inclusion of positive con-trol antibody (i.e., serum from a convalescent SARS patient)and human serum negative for SARS-CoV-specific antibody.
3. Results3.1. Characterization of mAbs to SARS-CoVTwenty-six B cell hybridoma cell lines were made thatproduced mAbs reactive to SARS-CoV by ELISA. On thebasis of ELISA reactivity of the mAbs reactive for SARS-CoV-infected Vero E6 cell lysate (positive control, P) and asimilarly prepared uninfected Vero E6 cell lysate (negativecontrol, N), nine B cell hybridomas with P/N ratios≥3 weresubcloned and expanded, and the mAbs were characterizedfurther. Four of the nine mAbs were of the subclass IgM,four were of subclass IgG2a and one was of subclass IgG1(Table 1).3.2. Specificity of the mAbsThe specificities of the nine mAbs were determined by im-munofluorescence antibody staining of transfected Vero cellsexpressing SARS-CoV E, M, N or S proteins. The resultsshowed that two mAbs reacted against the M protein, fivereacted against the S protein, one reacted weakly against theE protein, and one reacted weakly against N protein. A repre-sentative IFA using an S protein-specific mAb (mAb 341) andM protein-specific mAb (mAb 292) reactive to SARS-CoVinfected Vero E6 cells is shown inFig. 1. As expected, all themAbs examined reacted strongly with SARS-CoV-infectedVero E6 cells but not mock-infected cells.Radioimmunoprecipitation analysis confirmed mAbs re-active to the S (mAb 341, 534, 560, 240), M (mAb 283),and N (mAb 42) proteins (Fig. 2). In addition, anti-N proteinmAb specificity was confirmed by ELISA using recombinantN protein, and by Western Blot, using PEG-purified SARS-CoV-inactivated by gamma-irradiation (data not shown), andanti-S protein mAb specificity was confirmed by reactivity
R .A .Tripp et al ./ Journal of Virological Methods 128 (2005) 21–2825Fig. 1. Immunofluorescence staining (IFA) of SARS-CoV infected Vero E6 cells. A representative IFA is shown for an S protein-specific mAb (mAb 341) andM protein-specific mAb (mAb 292). Cell staining was visualized at 40×and 100×using a Zeiss Axioskop microscope with an Axiovert BlueH 485 nm filterand an RT Color Spot digital camera.
Fig. 3. Schematic diagram of the various S glycoprotein fragments synthe-sized. The top box represents the whole length of SARS-CoV S glycoprotein(aa 1–1255) and the relative sizes of the selected S protein fragments areshown.against recombinant S protein by ELISA and by Western Blotanalysis, using inactivated SARS-CoV, full-length soluble Sprotein, and S protein fragments.The S protein fragments contained amino acids (aa)1–269, 1–350, 1–490, 1–510, 1–1190 and 270–510 (Fig. 3).The development and characterization of these fragments hasbeen described previously (Babcock et al., 2004). The re-activity patterns against the S protein fragments indicatedthe region recognized by these antibodies. S protein mAbs154C and 560C reacted to fragments S350,S490,S510,S1190,and S270–510indicating these antibodies recognize an epitopewithin amino acids 270–350. Monoclonal antibodies 240C,341C, 534C reacted to S protein fragments S510,S1190, andS270–510,suggesting that these antibodies recognized a secondepitope within amino acids 490–510 (Table 2). A represen-tative Western Blot for a neutralizing anti-S protein mono-clonal, 341C (Fig. 4a), and a non-neutralizing anti-S proteinmonoclonal, 560C (Fig. 4b), are shown.3.3. Neutralization of SARS-CoVFive of the nine mAbs were reactive to the S protein, andmAbs 341C and 534C neutralized SARS-CoV infection ofVero E6 cells. Of note, both neutralizing antibodies werereactive to the same epitope (i.e., aa 490–510), while non-neutralizing mAbs, (with the exception of antibody 240C),recognized a second epitope, i.e., aa 270–350 (Table 2). Thereactivity patterns combined with two functional assays, virusneutralization and receptor blocking results (data not shown),were consistent with described previously sites on the S pro-tein involved in binding to ACE-2 and in virus neutralization.
Fig. 4. Epitope mapping of neutralizing and non-neutralizing anti-S mAbs.The epitopes recognized by the anti-S protein mAbs were determined byWestern Blot with C-terminally truncated S proteins covering the regionbetween amino acids 269 and 1190 of the S protein. S1190, aa 1–1190; S510,aa 1–510; S490, aa 1–490; S350, aa 1–350; S270–510, aa 270–510; S269,aa 1–269. A representative Western Blot for a neutralizing anti-S proteinmonoclonal, 341C (A), a non-neutralizing anti-S protein monoclonal, 560C(B), SARS convalescent-phase sera (C) and anti-(His)6antibody (D) areshown.4. DiscussionThe mAbs described in this study will facilitate detectionof the structural proteins of SARS-CoV, provide a means todiagnose infection, and aid in the study of the biology ofSARS-CoV infection and disease pathogenesis. The abilityto detect specific proteins has proven useful for assessingprotein-specific antibody responses and for developing diag-nostic assays. On the basis of results from studies of othercoronavirus infections in animals (Daniel and Talbot, 1990;De Diego et al., 1992; Ignjatovic and Galli, 1993; Kusters etal., 1989; Saif, 1993) and on the serum antibody response ofpatients with SARS (Che et al., 2003), the antibody responseto SARS-CoV appears to be dominated by anti-N protein andanti-S protein antibodies. Thus, mAbs reactive to N or S pro-teins can be used in antibody capture assays and provide akey reagent for high-quality IgM and IgA antibody assays. Inaddition, the anti-S protein mAbs can be used to study virusneutralization, and to better understand the function of thetwo epitopes against which they react.There is a well-recognized need for mAbs reactive toSARS-CoV proteins. A recent study described the first neu-tralizing murine mAbs reactive to SARS-CoV (Berry et al.2004). In that study, two of five mAbs reactive to S pro-tein neutralized SARS-CoV infection of Vero cells, one mAbreacted to N protein, and eleven mAbs had undeterminedspecificity (Berry et al., 2004). In addition, three groups havedescribed neutralizing human mAbs reactive against SARS-CoV (Sui et al., 2004; Traggiai et al., 2004; Zhou et al., 2004).In this study, the authors show the availability of murinemAbs reactive to the four structural proteins of SARS-CoVand two S protein epitopes that fill an important gap in im-munological tools needed to further explore the biology ofSARS-CoV infection and pathogenesis of disease. The mAbsshould help the development of diagnostic assays and stud-ies examining the biology of infection and pathogenesis ofdisease.
refer
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