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MAJOR ARTICLE Fully Human Monoclonal Antibody Directed to Proteolytic Cleavage Site in Severe Acute Respiratory Syndrome SARS Coronavirus S Protein Neutralizes the Virus in a Rhesus Macaque SARS Model Tohru Miyoshi Akiyama 1 Isao Ishida 2 Masaya Fukushi 1 Keina Yamaguchi 6 Yusuke Matsuoka 1 Takashi Ishihara 6 Masayoshi Tsukahara 6 Seisuke Hatakeyama 1 Norikazu Itoh 1 Aki Morisawa 1 Yoshiyuki Yoshinaka 3 Naoki Yamamoto 4 Zhang Lianfeng 7 Qin Chuan 7 Teruo Kirikae 1 and Takehiko Sasazuki 5 1 Department of Infectious Diseases National Center for Global Health and Medicine 1 21 1 Toyama Shinjuku ku 2 Frontier Laboratory Kyowa Hakko Kirin 1 6 1 Otemachi Chiyoda ku 3 Department of Molecular Virology Bio Response Graduate School Tokyo Medical and Dental University 1 5 45 Yushima Bunkyo 4 AIDS Research Center National Institute of Infectious Diseases and 5 National Center for Global Health and Medicine 1 21 1 Toyama Shinjuku ku Tokyo and 6 Bio Process Research and Development Laboratories Production Division Kyowa Hakko Kirin 100 1 Hagiwara cho Takasaki shi Gunma Japan and 7 Institute of Laboratory Animal Science Chinese Academy of Medical Sciences and Peking Union Medical College Beijing PR China Background There is still no effective method to prevent or treat severe acute respiratory syndrome SARS which is caused by SARS coronavirus CoV In the present study we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS CoV in vitro in a Rhesus macaque model of SARS Methods The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS Results 5H10 which recognized the same epitope that is also a cleavage site critical for the entry of SARS CoV into host cells inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route In addition we analyzed the mode of action of 5H10 and the results suggested that 5H10 inhibited fusion between the virus envelope and hostcell membrane 5H10 has potential for use in prevention and treatment of SARS if it reemerges Conclusions This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner Severe acute respiratory syndrome SARS has been observed inC2430 countries affecting 8000 persons and resulting in death in C2410 of cases 1 The disease is caused by the newly identified SARS coronavirus CoV 1 3 Although bats carry viruses similar to SARS CoV 4 its natural host has not yet been identified and no vaccine or drug treatment has yet been approved Thus there is still a risk of the reemergence of SARS in the human population and it is necessary to develop effective measures to combat this disease SARS CoV enters host cells by a mechanism involv ing the interaction between its spike S protein and human angiotensin converting enzyme 2 ACE2 ex pressed on host cells in the lung 5 During entry S is cleavedintothe N terminalS1regionandC terminalS2 region S1 mediates binding to ACE2 and S2 mediates fusion of viral and host cell membranes 6 7 A region named S791 in this study corresponding to amino acid Received 23 June 2010 accepted 31 August 2010 Potential conflicts of interest none reported Correspondence Tohru Miyoshi Akiyama PhD Dept of Infectious Diseases Research Institute National Center for Global Health and Medicine 1 21 1 Toyama Shinjuku ku Tokyo 162 8655 Japan takiyam ri ncgm go jp The Journal of Infectious Diseases 2011 203 1574 81 C211 The Author 2011 Published by Oxford University Press on behalf of the Infectious Diseases Society of America All rights reserved For Permissions please e mail journals permissions 0022 1899 print 1537 6613 online 2011 20311 0011 14 00 DOI 10 1093 infdis jir084 1574 d JID 2011 203 1 June d Miyoshi Akiyama et al at University of New Orleans on June 2 2015 http jid oxfordjournals org Downloaded from positions 620 to 900 of S contains cleavage sites for S1 and S2 domains 8 S791 also contains an epitope recognized by serum samples from convalescing patients with SARS in Vietnam 9 The Sproteinalsoplaysanimportantroleininducingprotective immunity 10 12 Antibodies targeting the S1 domain how ever also induced antibody dependent enhancement of SARS CoV entry into the host cells when the amino acid sequences of S proteins were changed 13 14 Monoclonal antibodies against SARS CoV have been generated in both human and murine backgrounds 15 17 Some of the monoclonal antibodies mAbs were tested for passive immuni zation in mice ferrets and hamsters and showed the ability to prevent or limit infection 17 20 Because all in vivo evaluations of the mAbs have been performed in models lacking detectable clinical symptoms and disease it isdifficulttoassesswhetherthe antibodies tested would actually provide reasonable protection in humans The Rhesus macaque is one of the most valuable tools for evaluating the efficacy of antibodies against SARS CoV because this model showed virus propagation immunological response and pathological changes in the lungs of infected animals 21 22 In the present study we produced a fully human monoclonal antibody 5H10 by immunization of KM mice which produce human antibodies 23 with fragments derived from the SARS S protein prepared in Escherichia coli but not SARS CoV to obtain human antibodies capable of neutralizing the virus We examinedthe neutralizingactivityof 5H10against SARS CoVin vivo in the Rhesus macaque SARS model MATERIALS AND METHODS Expression and Purification of Recombinant S Proteins and Antibodies Against Them All recombinant proteins Supplemental Fig 1 were expressed as His tagged proteins using the TAGZyme pQE2 vector Qiagen and were affinity purified using the His tag The His tags in the proteins were removed enzymatically as described elsewhere 24 and used for vaccination of animals All animal experiments were performed in accordance with the guidelines of the Ethics Review Committees of Animal Experiments of International Medical Center of Japan Aliquots of 100 lgof purified proteins animal were used for vaccination of rabbits or KM mice 23 transgenic animals that produce fully human antibodies with Freund s adjuvant Difco Laboratories To produce human monoclonal antibodies the spleen cells from KM mice vaccinated with the S791 fragment were fused with the mouse myeloma cell line SP2 O Ag14 and hybridomas were screened for theirability tobindtorecombinant S791protein by conventional enzyme linked immunosorbent assay as described elsewhere 23 Mass production of 5H10 was performed in fed batch culture of CHO cells as described elsewhere 25 Epitope mapping of mAbs was performed using a set of overlapping peptides 70 purity spanning the entire sequence of the S791 protein each consisting of 15 amino acids overlapping the next peptide by 5 amino acids as described elsewhere 24 Neutralization of SARS CoV In Vitro by 5H10 All experiments using SARS CoV were performed in BSL3 level facilities The neutralizing activities of mAbs against SARS CoV Frankfurt01 were determined by plaque reduction assay Monolayers of Vero E6 cells were grown in Dulbecco s Modified Eagle Medium DMEM supplemented with 5 fetal calf serum in 6 well plates at 37C176C with 5 carbon dioxide Samples 100 mL of 5H10 were incubated with an equal volume of SARS CoV suspension containing 200 plaque forming units of virus in a water bath at 37C176C for 1 h Samples of the incubated mixture were used to infect Vero E6 monolayers After adsorption for 1 h the cultures were overlaid with 1 methylcellulose containing media and incubated for 6 8 days at 37C176C The cells were then fixed with 20 formalin stained with 0 1 crystal violet in 0 1 M citric acid and irradiated with UV light for 1 h After drying the numbers of virus plaques were counted The neutralizing titer was defined as the reciprocal dilution of serum causing a 50 reduction in the plaque count compared with the negative control In the neutralization assay against SARS CoV PUMC01 cell monolayers were stained with 100 lLof0 15 neutralred Sigma in DMEM for 1 h at 37C176C After washing 100 lLofacid alcohol 1 acetic acid in 50 ethanol was added to each well After incubation for 30min at room temperature the absorbance of neutral red stained plates was read at 450 nm Cell Cell Fusion Assay Mediated by Interaction Between SARS S and Human ACE2 The full length human ace2 gene in pcDNA3 1 2 kindly provided by Dr H Choe Harvard Medical School the human codon optimized spike gene of SARS CoV in the VRC8304 vec tor kindly provided by Dr G J Nabel National Institutes of Health and pGL4 35 encoding luciferase pACT MyoD and pBIND Idplasmids Promega wereusedinthefusionassay The human ace2 gene and the spike gene of SARS CoV were inserted into pIRES2 DdRed Express2 and pIRES2 AcGFP1 Clontech respectively and used in the syncytium formation assay The spike gene and pBIND Id plasmids were transfected into HEK293T cells with Lipofectamine LTX Reagent Invitrogen in one group The human ace2 gene pACT MyoD and pGL4 35 were transfected into the other HE293T cell group Two days after transfection HEK293T cells expressing S protein were de tached with trypsin EDTA solution Sigma or EDTA solution HEK293T cells expressing ACE2 protein were detached with EDTA solution The treatments with anti SARS antibody were performed before and after detachment These cells were mixed and cocultured for 1 day at 37C176C The cells were harvested with lysis buffer and centrifuged The cell lysates were then mixed with the substrate PicaGene Toyo Ink and luciferase activity was measured using a plate reader Infinite F200 Tecan Fully Human Antibody Against SARS CoV d JID 2011 203 1 June d 1575 at University of New Orleans on June 2 2015 http jid oxfordjournals org Downloaded from In Western blot analysis HEK293T cells expressing S protein were treated in the presence or absence of 5H10 Cells were de tached using trypsin EDTA or EDTA solution After antibody treatment and detachment these cells were lysed with radio immunoprecipitation assay solution 0 1 sodium dodecyl sul fate 0 5 sodium deoxycholate 1 Nonidet P 40 150 mM sodium chloride 50 mM Tris HCl pH 8 0 The cell lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto Immun Blot P poly vinylidene difluoride membranes Bio Rad Laboratories The membranes were incubated with a mixture of in house anti S1 N S1 C S791 and S2 antibodies described above and anti rabbit IgG horseradish peroxidase GE Healthcare After washing the membranes were incubated with SuperSignal West Substrate Thermo Scientific according to the manufacturers instructions Syncytium Formation Assay and Inhibition by Anti SARS Antibodies The spike gene in pIRES2 AcGFP1 and ace2 gene in pIRES2 DdRed Express2 were transfected into HEK293T cells sepa rately Two days after transfection HEK293T cells expressing S protein were detached with trypsin EDTA solution or EDTA solution On the other hand HEK293T cells expressing ACE2 protein were detached with EDTA solution These cells were mixed and cocultured for 6 h at 37C176C and observed with an Olympus IX 70 microscope Evaluation of 5H10 in Rhesus Macaques In Vivo All the procedures were approved by the Institute of Animal Use and Care Committee of the Institute of Laboratory Animal Science Peking Union Medical College The sham administra tion control human IgG administration and 5H10 adminis tration groups of Rhesus macaques 3 4 years of age were used for experimental infection with SARS CoV PUMC01 by spraying of the virus median culture tissue infective dose 1 3 10 26 into the nasal cavity of the animals separately as described elsewhere 26 The antibodywasadministeredintravenously1 h before virus infection and on days 1 and 3 after infection at a dose of 12 5 mg kg total dose 37 5 mg kg Animals were sacrificed 6 days after infection and lung tissue samples were collected Organs were grossly examined and were photographed to record lung damage Haematoxylin and eosin staining of the tissue sections detection of viral antigens and determination of SARS CoV load by reverse transcription polymerase chain reaction RT PCR were performed as de scribed elsewhere 22 Preliminary safety tests of 5H10 in the Rhesus model The 3 aforementioned groups of Rhesus macaques without SARS CoV infection were used for the tests The antibodies were injected intravenously 3 times at 24 h intervals at a dose of 12 5 mg kg total dose 37 5 mg kg Full evaluation results are available upon request RESULTS Recombinant Spike Fragments To prepare antigen without using hazardous SARS CoV we used recombinant protein technologies using only genomic sequence information The gene encoding SARS S protein was obtained from a commercial service and used for the expression of 4 S fragments separately Supplemental Fig 1a Fragmen tation of the S protein was based on the functional domain No function has yet been assigned to S1 N and S1 C is the domain responsible for ACE2 binding 5 S791 and S2 contain cleavage sites for virus entry after binding of ACE2 10 and heptad repeats for trimerization of S supporting the interaction ofSwithACE2 7 respectively These fragmentswereexpressed in E coli as His tagged proteins and were purified to homoge neity using the tag To avoid raising antibodies against the tag the His tag was removed by enzymatic cleavage N terminal amino acid sequence analysis confirmed cleavage of the His tag at the expected amino acid position The S fragments obtained were evaluated for the ability to induceneutralizing antibodiesinrabbits SupplementalFig 1b Although all fragments induced antibody responses when used as antigens for vaccination of rabbits only the S791 frag ment induced an antibody that neutralized SARS CoV in vitro Supplemental Fig 1c Generation of Fully Human mAb Against SARS CoV Using the S791 Fragment On the basis of the ability to induce neutralizing antibodies the S791 fragment was used to vaccinate KM mice to obtain fully human mAb Three mAbs 65B3 63B10 and 5H10 reactive to the S791 fragment were chosen to characterize the epitopes recognized by them and the ability to neutralize SARS CoV in vitro 65B3 and 63B10 reacted with a peptide corre sponding to amino acid positions 731 to 745 of the S protein CANLLLQYGSFCTQL In contrast 5H10 reacted with a peptide corresponding to amino acid positions 791 to 805 of the S protein PLKPTKRSFIEDLLF data not shown The latter peptide was identical to that recognized by the serum samples from convalescing patients with SARS in Vietnam 9 Furthermore the amino acid R797 is the trypsin pro teolytic site to enhance fusion of the virus 8 Among the 3 mAbs only 5H10 showed in vitro SARS CoV neutralizing activity and the 50 effective concentration was 5 lg mL Supplemental Fig 1d These results indicated that 5H10 recognizing the proteolytic site of the S protein is a potent fully human neutralizing mAb against SARS CoV Inhibition of S ACE2 Mediated Cell Fusion by 5H10 To examine the mechanism of SARS CoV neutralization by 5H10 we analyzed cell cell fusion between S expressing HEK293 cells and ACE2 expressing HEK293 cells using 1576 d JID 2011 203 1 June d Miyoshi Akiyama et al at University of New Orleans on June 2 2015 http jid oxfordjournals org Downloaded from a reporter system based on the mammalian 2 hybrid system After cell cell fusion mediated by S and ACE2 the dual luciferase reporter system was activated and the amount of cell cell fusion could be quantified Because treatment with trypsin enhances cell cell fusion via cleavage of a site located in the S791 region 10 27 fusion efficiency of the cells treated with EDTA only was compared with that of cells treated with EDTA and trypsin in the presence or absence of 5H10 Figure 1a When S expressing cells were detached from culture dishes by treatment with trypsin in addition to EDTA cellular fusion reflected by relative fluorescence was significantly in creased in comparison with detachment by EDTA only Cell fusion was strongly inhibited by 5H10 but not human IgG when cells were treated with the antibodies after detachment from the dishes In contrast 5H10 did not show inhibition of cell fusion when added before detachment from the dishes We also analyzed cell cell fusion mediated by S ACE2 in teraction using confocal laser microscopy Figure 1b S expressing cells and ACE2 expressing cells were labeled with green fluorescent protein and DsRed respectively Although S expressing cells and ACE2 expressing cells were partly fused even after detachment with EDTA only syncytium formation was strongly enhanced by treatment with trypsin Addition of 5H10 to the cell mixtures inhibited syncytium formation at least in part in those treated with or without trypsin We also analyzed the cleavage of S by trypsin in the presence or absence of 5H10 Figure 1c By trypsin treatment S was cleaved generating an additional fragment of ca 90 kDa indicated by an arrow Addition of 5H10 during trypsin treatment did not significantly affect generation of the 90 kDa fragment Furthermore 5H10 reacted with the 90 kDa fragment as de termined by Western blot data not shown suggesting that uncleaved S is not necessary for recognition by 5H10 These results indicated that 5H10 inhibited the cell cell fusion step but not cleavage of S or virus propagation in the virus replication cycle SARS CoV Neutralizing Activity In Vivo Using a Rhesus Macaque Model Before analyzing the efficacy of 5H10 in an animal model we examinedwhether 5H10has in vitro neutralizing activityagainst a SARS CoV strain isolated in China PUMC01 The results indicated that 5H10 neutralized the PUCM01 strain data not shown To analyze the efficacy of 5H10 to protect against SARS CoV infection in Rhesus macaques 9 animals were separated into 3 groups 3 animals group consisting of sham administration control IgG administration and 5H10 administration All of the animals were infected with the virus PUMC01 strain by spraying into the nasal cavity The antibody was injected in travenously 1 h before virus infection and on days 1 and 3 after infection at a dose of 12 5 mg kg total dose 37 5 mg kg The dose of 12 5 mg kg was aimed to archive 10 fold 50 inhibition Figure 1 Inhibitory effects of 5H10 on cell cell fusion mediated by S ACE2 interaction A Effects of 5H10 in cell cell fusion analysis using a dual reporter system Fusion of S expressing 293 cells and human ACE2 expressing 293 cells was quantified as described in Materials and Methods Cells were detached with or without trypsin in addition to EDTA The antibody treatments were performed before or after detachment as indicated B Effects of 5H10 on syncytium formation by S expressing cells and human ACE2 expressing cells Syncytium formation of S and ACE2 expressing cells labeled with GFP and DsRed respectively was visualized by confocal laser microscopy Cells were detached with or without trypsin in addition to EDTA then mixed and 5H10 was added to the cultures as indicated C Western blotting analysis to determine the effects of 5H10 on S cleavage by trypsin treatment S expressing 293T cells were treated with trypsin or EDTA in the presence or absence of 5H10 3 mg mL as indicated After lysis of the cells Western blotting using anti S antibody mixture was performed Control 293T cells which did not harbor the spike gene were used as a negative control Fully Human Antibody Against SARS CoV d JID 2011 203 1 June d 1577 at University of New Orleans on June 2 2015 http jid oxfordjournals org Downloaded from concentration in the blood according to the results of in vitro SARS CoV neutralization assay Gross pathological character ization of the lung on day 6 in animals administered 5H10 showed significant reduction of the severity of damage in all