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Brian Abstract for tation were proteins the terized K 1 stranded Department 0166 0934 doi 10 1016 j jviromet 2005 03 021 Journal of Virological Methods 128 2005 21 28 Monoclonal antibodies to SARS associated coronavirus SARS CoV Identification of neutralizing and antibodies reactive to S N M and E viral proteins Ralph A Tripp a 1 Lia M Haynes a Deborah Moore a Barbara Anderson a Azaibi Tamin a H Harcourt a Les P Jones a 1 Mamadi Yilla a Gregory J Babcock c Thomas Greenough c Donna M Ambrosino c Rene Alvarez a 1 Justin Callaway d Sheana Cavitt d Kurt Kamrud e Harold Alterson e Jonathan Smith e Jennifer L Harcourt a Congrong Miao a Raj Razdan a James A Comer b Pierre E Rollin b Thomas G Ksiazek b Anthony Sanchez b Paul A Rota a William J Bellini a Larry J Anderson a a National Centers for Infectious Diseases Division of Viral and Rickettsial Diseases Respiratory and Enteric Virus Branch Centers for Disease Control and Prevention 1600 Clifton Rd NE Mailstop G 09 Atlanta GA 30333 USA b National Center for Infectious Diseases Division of Viral and Rickettsial Diseases Special Pathogens Branch Centers for Disease Control and Prevention Atlanta GA 30333 USA c Massachusetts Biologic Laboratories University of Massachusetts Medical School Jamaica Plain MA 02130 USA d Furman University Greenville SC 29613 USA e AlphaVax Inc Research Triangle Park NC 27709 USA Received 29 November 2004 received in revised form 23 March 2005 accepted 23 March 2005 Available online 10 May 2005 Monoclonal antibodies Mabs against the Urbani strain of the SARS associated reactivity to SARS CoV and SARS CoV S N M and E proteins using immunofluorescence Western Blot and microneutralization assays chosen for detailed characterization Five mAbs reacted against the S Two of five S protein mAbs neutralized SARS CoV infection of Vero S protein While two of the three non neutralizing antibodies recognized should prove useful for developing SARS CoV diagnostic assays and 2005 Elsevier B V All rights reserved eywords SARS coronavirus Monoclonal antibody Immunoassay Epitope Neutralizing Introduction Coronaviruses CoVs are large enveloped positive RNA viruses that cause a variety of illnesses in Corresponding author Tel 1 404 639 4004 fax 1 404 639 1307 E mail address loh5 cdc gov L M Haynes 1 Present address University of Georgia College of Veterinary Medicine of Infectious Diseases Athens GA 30602 USA humans 1998 et one global w has Hoek see front matter 2005 Elsevier B V All rights reserved coronavirus SARS CoV were developed and characterized enzyme linked immunoabsorbent ELISA radioimmunoprecipi Twenty six mAbs were reactive to SARS CoV by ELISA and nine protein two against the M protein and one each against the N and E E6 cells and reacted to an epitope within amino acids 490 510 in at second epitope within amino acids 270 350 The mAbs charac for studying the biology of infection and pathogenesis of disease and animals Lai 1990 Lavi et al 1999 Perlman Snijder and Horzinek 1993 Snijder et al 1993 Zhou al 2004 Most human coronaviruses HCoVs fall into of two serotypes OC43 like and 229E like however the outbreak of severe acute respiratory syndrome SARS as quickly linked to infection with a novel CoV SARS CoV Ksiazek et al 2003 Peiris et al 2003 and HCoV NL63 been recognized recently as a human pathogen van der et al 2004 The CoV genome encodes numerous non 22 gical structural ing en HE teins The for protecti tropism forms cleocapsid is cally ha Human to ond associated disease et usually ease 15 age Ksiazek Although June ha been cember ing pandemic reagents CoV were include proteins tralize 2 2 1 3 Control inacti its the diation 2 2 sential plemented Hyclone The to col 1980 for CoV tion 2 3 guidelines tee mice toneally CoV follo later 2 2 2 tribromoethanol munization cells 1000 tured 1984 mented and R A Tripp et al Journal of Virolo proteins and four or five structural proteins includ the spike S nucleocapsid N membrane M small velope E and in some strains a hemagglutinin esterase protein Lai 1990 SARS CoV has four structural pro the S N M and E proteins that have various functions S protein forms spikes on the virion surface and is crucial viral attachment and entry into the host cell It also induces ve immunity and is associated with host range tissue and virulence Sanchez et al 1999 The N protein the nucleocapsid the M protein interacts with the nu and forms the internal viral core and the E protein associated with the viral envelope SARS CoV is geneti distinct from previously described coronaviruses which ve been placed into three antigenic groups I II and III coronaviruses i e 229E like and OC43 like belong groups I and II respectively and are recognized as the sec most common cause of upper respiratory disease but are infrequently with serious lower respiratory tract El Sahly et al 2000 Hendley et al 1972 Makela al 1998 Falsey et al 2002 However SARS CoV is associated with serious lower respiratory tract dis having a fatality rate as ranging between 10 and that may be as high as 50 in patients 60 years of Drosten et al 2003 Enserink 2003 Holmes 2003 et al 2003 Poutanen et al 2003 Rota et al 2003 the global spread of SARS CoV was stopped in 2003 six instances of laboratory acquired infections ve been confirmed and a cluster of sporadic cases have detected in Guangdong Province China between De 2003 and April 2004 Liang et al 2004 demonstrat the potential for SARS to re emerge and possibly become Anticipating the need for improved immunological to aid identification and characterization of SARS monoclonal antibodies mAbs to SARS CoV proteins produced This report describes nine such mAbs that antibodies reactive against each of the four structural including two S protein reactive antibodies that neu SARS CoV Materials and methods Biosafety All work with live SARS CoV was done in biosafety level BSL 3 containment laboratories at the Centers for Disease and Prevention Atlanta Georgia SARS CoV was vated by 60 Co gamma irradiation at 2 10 6 rad prior to use as an immunogen or as an antigen in an ELISA Within limits of detecting viable virus 2 10 6 rads gamma irra was sufficient to inactivate all infectivity Virus preparation Vero E6 cells were maintained in Dulbecco s minimal es media DMEM Invitrogen Corp Carlsbad CA sup HA ti against by acti control a control cells were termined Roche firmed cata 2 4 2 4 1 with SARS CoV histidine tagged metal chelate follo purified Methods 128 2005 21 28 with 10 heat inactivated fetal bovine sera FBS Logan UT and 2 mM l glutamine Invitrogen Urbani strain of SARS CoV was plaque purified grown stock titers in Vero E6 cells purified by polyethylene gly PEG precipitation as described previously Kiley et al and frozen at 70 C until use Viral antigen used ELISA was prepared by detergent extraction of SARS infected Vero E6 cells and subsequent gamma irradia Ksiazek et al 2003 B cell hybridoma production Immunizations were performed in accordance with the of the Institutional Animal Care and Use Commit Female 4 6 week old specific pathogen free BALB c Jackson Laboratories Bar Harbor ME were intraperi i p administered 200H9262l of PEG purified SARS inactivated by gamma irradiation and diluted in PBS wed by two similar immunizations 3 and 6 weeks Mice were euthanized by exsanguination under Avertin anesthesia 3 days after the last im and their spleens were removed Splenocytes were fused with non secretor SP2 0 myeloma at a 2 1 ratio using 50 polyethylene glycol 1000 PEG Sigma Chemical Company St Louis MO and cul in 24 well plates as described previously Reimer et al B cell hybridomas were selected using DMEM supple with 10 heat inactivated FBS 2 mM l glutamine 1 sodium hypoxanthine aminopterin and thymidine T Invitrogen and culture fluids were screened for reac vity against a SARS CoV infected Vero E6 cell lysate and a similarly prepared uninfected Vero E6 cell lysate ELISA The mean optical density OD of the mAbs re ve to SARS CoV infected Vero E6 cell lysate positive P was divided by the mean OD of mAbs reactive to similarly prepared uninfected Vero E6 cell lysate negative N and were termed P N ratios B cell hybridoma from wells that gave positive to negative P N ratios 3 were cloned by limiting dilution Hybridomas cell lines grown in DMEM supplemented with 10 FBS Monoclonal immunoglobulin class and isotype were de by use of the IsoStrip mouse mAb isotyping kit Diagnostics Corporation Indianapolis IN and con for isotype by ELISA Amersham Biosciences Pis way NJ as described by the manufacturer Recombinant proteins Recombinant SARS CoV N protein Escherichia coli BL21 cells Invitrogen were transformed an inducible pET vector expressing 6 histidine tagged N protein Invitrogen and expressed 6 SARS CoV N protein was purified by chromatography ProBond resin Invitrogen wing the manufacturer s recommended protocols The 6 histidine tagged SARS CoV N protein was gical eluted 10 free ate PBS ti 2 4 2 protein ments and thesized Coomassie con tection tein synthetic 2 5 M of the for a The specific F tion the data and M v Eco PCR tor transcribed boMax manuf 2 6 e pressing tion media were 50 spot were fix perature immunofluorescence human chick OR VEE E sho 2 7 E6 mock infected SARS CoV et 2 8 inacti E6 described protocol SARS CoV control antigen 2 9 R A Tripp et al Journal of Virolo in lysis buffer 100 mM sodium phosphate monobasic mM Tris HCl 300 mM NaCl in PBS containing EDTA proteinase inhibitor pH 8 0 adjusted to pH 4 5 The elu was immediately adjusted to pH 7 5 and dialyzed against The purified N protein was tested by ELISA for reac vity to hyperimmune mouse sera and anti SARS N mAb Synthetic SARS S protein fragments The full length soluble portion of the SARS CoV S amino acids 1 1190 S 1190 and S protein frag containing amino acids 1 269 1 350 1 490 1 510 270 510 S 269 S 350 S 490 S 510 S 270 510 were syn expressed and purified as described previously Babcock et al 2004 Protein fragments were analyzed by staining and by Western Blot using human SARS valescent serum or mouse anti synthetic S protein for de The convalescent serum recognized all the S pro fragments with the exception of S269 while the anti S protein recognized all the S protein fragments Construction of replicons expressing SARS CoV S N and E proteins RNA was extracted from a preparation of the Urbani strain SARS CoV in Trizol LS reagent Invitrogen following manufacturer s protocol Reverse transcription reactions generation of SARS CoV cDNA were performed with Super Script III kit Invitrogen using random hexamers SARS CoV cDNA was PCR amplified with the gene primer pairs using Platinum Taq Hifi Invitrogen ollowing 18 cycles of amplification a sample of each reac was analyzed by gel electrophoresis and fragments of appropriate size were noted for each of the four genes not shown The gene PCR products were sequenced the expected sequences were confirmed The SARS CoV N and E gene PCR products were cloned directly into the enezuelan equine encephalitis VEE replicon vector using RV and AscI restriction sites The SARS CoV S gene product was directly cloned into the VEE replicon vec using AscI and PmeI restriction sites RNA was in vitro from each SARS gene replicon DNA using a Ri T7 RNA transcription kit Promega following the acturer s protocol Venezuelan equine encephalitis VEE replicons xpressing SARS CoV S M N and E proteins Vero cells were electroporated with replicon RNA ex either SARS M N S or E genes After electropora the cells were incubated in media containing OptiPRO Invitrogen for 16 h at 37 C Electroporated cells mixed at a 2 3 1 ratio with non electroporated cells and H9262l of the cell mixture was placed on each spot of a 12 well slide VWR International Buffalo Grove IL The slides incubated at 37 C for 3 5 h washed with PBS and then ed with acetone and methanol 1 1 for 5 min at room tem V methionine c and ficity radiolabelled in 150 containing Mannheim ple Amersham radiolabeled SDS polyacrylamide 2 10 plo protein with S harv containing S temperature Methods 128 2005 21 28 23 The fixed monolayers were analyzed in an indirect assay IFA using SARS convalescent plasma as the primary antibody and Alexa Fluor 488 en anti human IgG H L Molecular Probes Eugene as the secondary antibody Cells electroporated with the replicon RNAs containing the SARS CoV S M N and genes were intensely fluorescent in these assays data not wn IFA IFA was performed on 2 paraformaldehyde fixed Vero cells infected with 1000 TCID 50 SARS CoV for 27 h Vero E6 or Vero E6 cell lines expressing S N M or E as described previously Harcourt al 2004 ELISA An indirect ELISA using detergent extracted SARS CoV vated by gamma irradiation or similarly prepared Vero cell lysate was used to determine the mAb reactivity as previously Ksiazek et al 2003 A similar ELISA was used to detect mAb reactivity to recombinant N protein untransformed E coli BL21 cell lysate S 1190 protein or angiotensin converting enzyme 2 control Radioimmunoprecipitation SARS CoV infected Vero E6 cells or mock infected ero E6 cells 2 5 10 5 were radiolabeled with 35 S ysteine 0 1 mCi ml ICN Irvine California reacted with individual mAbs to determine antigen speci as described previously Benaroch et al 1995 Briefly cells were collected by centrifugation and lysed lysis buffer 0 1 SDS 1 0 TritionX 100 1 NaDOC mM NaCl 10 mM Tris HCl pH 7 2 and 5 mM EDTA 1 mM PMSF and 100 mM Pefablock Boehringer Mannheim Germany Antigen antibody com xes were precipitated using Protein G sepharose beads Biosciences Uppsala Sweden and the bound immunoprecipitate was analyzed by 12 5 gel electrophoresis SDS PAGE MAb epitope mapping Immunoprecipitation and Western Blot analysis were em yed to determine the epitopes recognized by the anti S mAbs HEK 293 cells were transiently transfected DNA encoding various S protein fragments S 269 S 510 490 S 350 S 270 510 S 1190 and culture supernatants were ested 48 h post transfection Equal amounts of S protein supernatants were incubated with respective anti protein mAbs and protein G Sepharose for 2 h at room while shaking The beads were washed in PBS 24 gical and ing SDS P Millipore 1 anti His6 non f horseradish Jackson temperature an e blotting cubated temperature ducing The the PBS T anti S with the 2004 2 11 CoV as test antibody trols formed trol and 3 3 1 produced basis CoV similarly control subcloned further four 3 2 munofluorescence e sho reacted E sentati M infected mAbs V T MAbs Subclone 19C 42C 154C 240C 283C 341C 472C 534C 560C The protein by according R A Tripp et al Journal of Virolo protein was eluted in Laemmli sample buffer by boil the sample for 5 min Samples were resolved by 10 AGE and transferred to an Immobilon P membrane Billerica MA Membranes were washed using non fat dried milk in PBS Tween and incubated with mAb Invitrogen Blots were washed twice in 1 at dried milk in PBS 0 05 Tween 20 incubated with peroxidase conjugated HRP anti mouse IgG Laboratories Bar Harbor ME for 45 min at room and washed and bands were detected by use of enhanced chemiluminescence reagent Amersham and xposed to X Omat AR film for various times For Western 1 ml of each S protein fragment supernatant was in with Ni NTA agarose Invitrogen for 2 h at room Resin was washed with PBS mixed with re Laemmli buffer and separated by 10 SDS PAGE proteins were transferred to Immobilon P Millipore membranes were blocked using 5 non fat dried milk in ween overnight and incubated for 1 h with respective protein mAbs The blots were washed and incubated HRP conjugated anti mouse IgG After being washed bands were detected as described above Babcock et al Virus microneutralization assay The mAbs were tested for their ability to neutralize SARS infection of Vero E6 cells by microneutralization assay described Sui et al 2004 The microneutralization titer of antibody was the reciprocal of the highest dilution of test that showed inhibition in all triplicate wells Con were included for each microneutralization assay per and included back titration inclusion of positive con antibody i e serum from a convalescent SARS patient human serum negative for SARS CoV specific antibody acti and mAb N CoV anti S able 1 to SARS CoV Isotype IFA specificity a ELISA SARS CoV reactive b ELISA recombinant protein IgM M 8 2 1 3 IgM N 9 2 1 3 IgM S 9 57 0 IgG2a S 13 234 9 IgG1 M 3 7 1 3 IgG2a S 13 82 2 IgM E 4 3 1 3 IgG2a S 6 92 5 IgG2a S 18 717 5 a MAb specificity shown here was determined by IFA reactivity using Vero cells b Hybridoma supernatants were tested at a 1 80 dilutions for reactivity against SARS CoV positive to negative P N ratios are indicated c Hybridoma supernatants were tested at a 1 80 dilution for reactivity against the control by ELISA The positive to negative P N ratios are indicated d Hybridoma supernatants were tested at a 1 40 dilution for reactivity against recombinant ELISA The positive to negative P N ratios are indicated e Neutralizing antibody titers were measured as the reciprocal of the highest antibody to Sui et al 2004 Methods 128 2005 21 28 Results Characterization of mAbs to SARS CoV Twenty six B cell hybridoma cell lines were made that mAbs reactive to SARS CoV by ELISA On the of ELISA reactivity of the mAbs reactive for SARS infected Vero E6 cell lysate positive control P and a prepared uninfected Vero E6 cell lysate negative N nine B cell hybridomas with P N ratios 3 were and expanded and the mAbs were characterized Four of the nine mAbs were of the subclass IgM were of subclass IgG2a and one was of subclass IgG1 Table 1 Speci city of the mAbs The specificities of the nine mAbs were determined by im antibody staining of transfected Vero cells xpressing SARS CoV E M N or S proteins The results wed that two mAbs reacted against the M protein five against the S protein one reacted weakly against the protein and one reacted weakly against N protein A repre ve IFA using an S protein specific mAb mAb 341 and protein specific mAb mAb 292 reactive to SARS CoV Vero E6 cells is shown in Fig 1 As expected all the examined reacted strongly with SARS CoV infected ero E6 cells but not mock infected cells Radioimmunoprecipitation analysis confirmed mAbs re ve to the S mAb 341 534 560 240 M mAb 283 N mAb 42 proteins Fig 2 In addition anti N protein specificity was confirmed by ELISA using recombinant protein and by Western Blot using PEG purified SARS inactivated by gamma irradiation data not shown and protein mAb specificity was confirmed by reactivity S reactive c ELISA recombinant N protein reactive d Neutralizing titer e 1 1 10 2 1 10 1 1 10 1 1 10 1 1 10 1 1 1280 1 1 10 1 1 80 1 1 10 expressing SARS CoV S N M or E proteins infected cell lysate and uninfected Vero E6 cell lysate by ELISA full length soluble S protein aa 1 1190 and a similarly prepared ACE 2 N protein and a similarly prepared E coli BL21 control antigen dilution that completely inhibited Vero E6 cell lysis in triplicate wells R A Tripp et al Journal of Virological Methods 128 2005 21 28 25 Fig M and Fig with S anti nucleocapsid in 1 Immunofluorescence staining IFA of SARS CoV infected Vero E6 cells A protein specific mAb mAb 292 Cell staining was visualized at 40 and 100 an RT Color Spot digital camera 2 Radioimmunoprecipitation of mAbs with SARS CoV lysate SARS CoV infected 35 S methionine cysteine and reacted with individual mAbs to determine antigen S mAbs 341 534 560 and 240 detected a protein with an apparent molecular N 42 mAbs detected proteins with an apparent molecular mass the uninfected Vero E6 lysate control The lack of signal with mAb 154 is likely representative IFA is shown for an S protein specific mAb mAb 341 and using a Zeiss Axioskop microscope with an Axiovert BlueH 485 nm filter Vero E6 cells or mock infected Vero E6 cells were radiolabeled specificity as described previously in Section 2 The anti SARS CoV mass of approximately 150 kDa while the anti matrix M 283 and of approximately 25 kDa and 45 kDa respectively No bands were detected due to the differential