Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex
Identifieur interne : 000302 ( Pmc/Checkpoint ); précédent : 000301; suivant : 000303Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex
Auteurs : Byoung Kwon Park [Corée du Sud] ; Sony Maharjan [Corée du Sud] ; Su In Lee [Corée du Sud] ; Jinsoo Kim [Corée du Sud] ; Joon-Yong Bae [Corée du Sud] ; Man-Seong Park [Corée du Sud] ; Hyung-Joo Kwon [Corée du Sud]Source :
- BMB Reports [ 1976-6696 ] ; 2019.
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future.
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DOI: 10.5483/BMBRep.2019.52.6.185
PubMed: 30355437
PubMed Central: 6605520
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex</title><author><name sortKey="Park, Byoung Kwon" sort="Park, Byoung Kwon" uniqKey="Park B" first="Byoung Kwon" last="Park">Byoung Kwon Park</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Maharjan, Sony" sort="Maharjan, Sony" uniqKey="Maharjan S" first="Sony" last="Maharjan">Sony Maharjan</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Lee, Su In" sort="Lee, Su In" uniqKey="Lee S" first="Su In" last="Lee">Su In Lee</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Kim, Jinsoo" sort="Kim, Jinsoo" uniqKey="Kim J" first="Jinsoo" last="Kim">Jinsoo Kim</name><affiliation wicri:level="1"><nlm:aff id="af2-bmb-52-397">Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Bae, Joon Yong" sort="Bae, Joon Yong" uniqKey="Bae J" first="Joon-Yong" last="Bae">Joon-Yong Bae</name><affiliation wicri:level="1"><nlm:aff id="af3-bmb-52-397">Department of Microbiology, College of Medicine, and the Institute for Viral Diseases, Korea University, Seoul 02841,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Park, Man Seong" sort="Park, Man Seong" uniqKey="Park M" first="Man-Seong" last="Park">Man-Seong Park</name><affiliation wicri:level="1"><nlm:aff id="af3-bmb-52-397">Department of Microbiology, College of Medicine, and the Institute for Viral Diseases, Korea University, Seoul 02841,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Kwon, Hyung Joo" sort="Kwon, Hyung Joo" uniqKey="Kwon H" first="Hyung-Joo" last="Kwon">Hyung-Joo Kwon</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-bmb-52-397">Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">30355437</idno><idno type="pmc">6605520</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605520</idno><idno type="RBID">PMC:6605520</idno><idno type="doi">10.5483/BMBRep.2019.52.6.185</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000A84</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A84</idno><idno type="wicri:Area/Pmc/Curation">000A84</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000A84</idno><idno type="wicri:Area/Pmc/Checkpoint">000302</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000302</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex</title><author><name sortKey="Park, Byoung Kwon" sort="Park, Byoung Kwon" uniqKey="Park B" first="Byoung Kwon" last="Park">Byoung Kwon Park</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Maharjan, Sony" sort="Maharjan, Sony" uniqKey="Maharjan S" first="Sony" last="Maharjan">Sony Maharjan</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Lee, Su In" sort="Lee, Su In" uniqKey="Lee S" first="Su In" last="Lee">Su In Lee</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Kim, Jinsoo" sort="Kim, Jinsoo" uniqKey="Kim J" first="Jinsoo" last="Kim">Jinsoo Kim</name><affiliation wicri:level="1"><nlm:aff id="af2-bmb-52-397">Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Bae, Joon Yong" sort="Bae, Joon Yong" uniqKey="Bae J" first="Joon-Yong" last="Bae">Joon-Yong Bae</name><affiliation wicri:level="1"><nlm:aff id="af3-bmb-52-397">Department of Microbiology, College of Medicine, and the Institute for Viral Diseases, Korea University, Seoul 02841,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Park, Man Seong" sort="Park, Man Seong" uniqKey="Park M" first="Man-Seong" last="Park">Man-Seong Park</name><affiliation wicri:level="1"><nlm:aff id="af3-bmb-52-397">Department of Microbiology, College of Medicine, and the Institute for Viral Diseases, Korea University, Seoul 02841,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Kwon, Hyung Joo" sort="Kwon, Hyung Joo" uniqKey="Kwon H" first="Hyung-Joo" last="Kwon">Hyung-Joo Kwon</name><affiliation wicri:level="1"><nlm:aff id="af1-bmb-52-397">Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-bmb-52-397">Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">BMB Reports</title><idno type="ISSN">1976-6696</idno><idno type="eISSN">1976-670X</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zumla, A" uniqKey="Zumla A">A Zumla</name></author><author><name sortKey="Chan, Jf" uniqKey="Chan J">JF Chan</name></author><author><name sortKey="Azhar, Ei" uniqKey="Azhar E">EI Azhar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L Du</name></author><author><name sortKey="Yang, Y" uniqKey="Yang Y">Y Yang</name></author><author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y Zhou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mackay, Im" uniqKey="Mackay I">IM Mackay</name></author><author><name sortKey="Arden, Ke" uniqKey="Arden K">KE 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<front><journal-meta><journal-id journal-id-type="nlm-ta">BMB Rep</journal-id><journal-id journal-id-type="iso-abbrev">BMB Rep</journal-id><journal-title-group><journal-title>BMB Reports</journal-title></journal-title-group><issn pub-type="ppub">1976-6696</issn><issn pub-type="epub">1976-670X</issn><publisher><publisher-name>Korean Society for Biochemistry and Molecular Biology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">30355437</article-id><article-id pub-id-type="pmc">6605520</article-id><article-id pub-id-type="doi">10.5483/BMBRep.2019.52.6.185</article-id><article-id pub-id-type="publisher-id">bmb-52-397</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group></article-categories><title-group><article-title>Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Park</surname><given-names>Byoung Kwon</given-names></name><xref ref-type="aff" rid="af1-bmb-52-397">1</xref></contrib><contrib contrib-type="author"><name><surname>Maharjan</surname><given-names>Sony</given-names></name><xref ref-type="aff" rid="af1-bmb-52-397">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Su In</given-names></name><xref ref-type="aff" rid="af1-bmb-52-397">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Jinsoo</given-names></name><xref ref-type="aff" rid="af2-bmb-52-397">2</xref></contrib><contrib contrib-type="author"><name><surname>Bae</surname><given-names>Joon-Yong</given-names></name><xref ref-type="aff" rid="af3-bmb-52-397">3</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Man-Seong</given-names></name><xref ref-type="aff" rid="af3-bmb-52-397">3</xref></contrib><contrib contrib-type="author"><name><surname>Kwon</surname><given-names>Hyung-Joo</given-names></name><xref ref-type="aff" rid="af1-bmb-52-397">1</xref><xref ref-type="aff" rid="af2-bmb-52-397">2</xref><xref rid="c1-bmb-52-397" ref-type="corresp">*</xref></contrib></contrib-group><aff id="af1-bmb-52-397"><label>1</label>Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></aff><aff id="af2-bmb-52-397"><label>2</label>Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252,<country>Korea</country></aff><aff id="af3-bmb-52-397"><label>3</label>Department of Microbiology, College of Medicine, and the Institute for Viral Diseases, Korea University, Seoul 02841,<country>Korea</country></aff><author-notes><corresp id="c1-bmb-52-397"><label>*</label>Corresponding author. Tel: +82-33-248-2635; Fax: +82-33-241-3640; E-mail: <email>hjookwon@hallym.ac.kr</email></corresp></author-notes><pub-date pub-type="ppub"><month>6</month><year>2019</year></pub-date><pub-date pub-type="epub"><day>30</day><month>6</month><year>2019</year></pub-date><volume>52</volume><issue>6</issue><fpage>397</fpage><lpage>402</lpage><history><date date-type="received"><day>08</day><month>8</month><year>2018</year></date><date date-type="rev-recd"><day>05</day><month>9</month><year>2018</year></date><date date-type="accepted"><day>11</day><month>10</month><year>2018</year></date></history><permissions><copyright-statement>Copyright © 2019 by the The Korean Society for Biochemistry and Molecular Biology</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/4.0">http://creativecommons.org/licenses/by-nc/4.0</ext-link>) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future.</p></abstract><kwd-group><kwd>B cell epitope</kwd><kwd>Lipoplex (O)</kwd><kwd>MERS-CoV</kwd><kwd>Monoclonal antibody</kwd><kwd>Spike protein</kwd></kwd-group></article-meta></front><floats-group><fig id="f1-bmb-52-397" orientation="portrait" position="float"><label>Fig. 1</label><caption><p>Production of the B cell epitope-specific antibody. (A) Schematic representation of the MERS-CoV S protein. The S protein consists of S1 and S2 subunits. RBD, receptor binding domain; FP, fusion peptide; HR1 and HR2, heptad repeat region 1 and 2; TM, transmembrane; CP, cytoplasmic tail. (B) Selection of the B cell epitope peptide sequence using the IEDB based on the epitope prediction, surface accessibility and antigenicity index. (C) ELISA analysis showing the generation of MERS-CoV Spike-492- or Spike-492 (L506F)-specific antibodies using sera. Sera were isolated from BALB/c mice immunized with a complex of B cell epitope peptide (Spike-492 or Spike-492 (L506F)) and Lipoplex (O).</p></caption><graphic xlink:href="bmb-52-397f1"></graphic></fig><fig id="f2-bmb-52-397" orientation="portrait" position="float"><label>Fig. 2</label><caption><p>Purification and characterization of anti-MERS-CoV Spike-492 (L506F) epitope-specific monoclonal antibody. (A) Purified 506-2G10G5 monoclonal antibody was analyzed by SDS-PAGE. R, reducing condition; NR, non-reducing conditions. (B) Isotype of the purified 506-2G10G5 monoclonal antibody was determined by ELISA. (C) The binding affinity of the 506-2G10G5 monoclonal antibody to the MERS-CoV Spike-492 (L506F) peptide was measured by ELISA, and the EC<sub>50</sub> value was evaluated with the Sigma Plot program. (D–F) MERS-CoV-infected and non-infected Vero cells lysates were treated with PBS (−) or PNGase F (+). The lysates were immunoblotted with the 506-2G10G5 monoclonal antibody (D). Western blot analysis using the 506-2G10G5 monoclonal antibody was performed following the immunoprecipitation of lysates with the 506-2G10G5 monoclonal antibody (E). Lysates were subjected to western blotting with an anti-β actin antibody (F). (G, H) Cross-reactivity of the monoclonal antibodies. 96-well immunoplates coated with the MERS-CoV Spike-492 (G) or Spike-492 (L506F) (H) peptides and incubated with either the 492-1G10E4E2 or 506-2G10G5 monoclonal antibody. The reactivity of the antibodies to each peptide was determined by ELISA assay.</p></caption><graphic xlink:href="bmb-52-397f2"></graphic></fig><fig id="f3-bmb-52-397" orientation="portrait" position="float"><label>Fig. 3</label><caption><p>Immunofluorescence assay and confocal microscopy for the detection of MERS-CoV-infected cells with the Spike-492 (L506F) monoclonal antibody. (A) Indirect immunofluorescence assay. A mixture of MERS-CoV-infected and non-infected Vero cells was stained with the 506-2G10G5 monoclonal antibody or normal mouse IgG, followed by incubation with the Alexa488-conjugated goat anti-mouse IgG antibody (green). Cells were counterstained with Hoechst 33258 for nuclear staining (blue). Images were taken using a fluorescence microscope. (B) Confocal microscopy. Vero cells were infected with MERS-CoV for 2 days. The cells were stained with the 506-2G10G5 monoclonal antibody or normal mouse IgG and then incubated with the Alexa488-conjugated goat anti-mouse IgG antibody (green). Nuclei were stained using Hoechst 33258 (blue). Scale bar, 10 μm.</p></caption><graphic xlink:href="bmb-52-397f3"></graphic></fig><fig id="f4-bmb-52-397" orientation="portrait" position="float"><label>Fig. 4</label><caption><p>Inhibition of MERS-CoV infection by the 492-1G10E4E2 and 506-2G10G5 monoclonal antibody. MERS-CoV was pre-incubated with two fold serially diluted normal mouse IgG, 492-1G10E4E2 or 506-2G10G5 monoclonal antibody for 30 min at 37°C. The virus-antibody mixture was added to the Vero cells and incubated for 1 h. After the incubation, the medium was replaced with DMEM/F12 containing 0.6% oxoid agar. The plaques were stained with crystal violet 4 days after infection. (A) A representative picture showing the plaque reduction assay. (B) Quatification of the plaque reduction assay against MERS-CoV after treatment with 100 μg/well to 0 μg/well of normal mouse IgG or 492-1G10E4E2 or 506-2G10G5 monoclonal antibody.</p></caption><graphic xlink:href="bmb-52-397f4"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Corée du Sud</li></country></list><tree><country name="Corée du Sud"><noRegion><name sortKey="Park, Byoung Kwon" sort="Park, Byoung Kwon" uniqKey="Park B" first="Byoung Kwon" last="Park">Byoung Kwon Park</name></noRegion><name sortKey="Bae, Joon Yong" sort="Bae, Joon Yong" uniqKey="Bae J" first="Joon-Yong" last="Bae">Joon-Yong Bae</name><name sortKey="Kim, Jinsoo" sort="Kim, Jinsoo" uniqKey="Kim J" first="Jinsoo" last="Kim">Jinsoo Kim</name><name sortKey="Kwon, Hyung Joo" sort="Kwon, Hyung Joo" uniqKey="Kwon H" first="Hyung-Joo" last="Kwon">Hyung-Joo Kwon</name><name sortKey="Kwon, Hyung Joo" sort="Kwon, Hyung Joo" uniqKey="Kwon H" first="Hyung-Joo" last="Kwon">Hyung-Joo Kwon</name><name sortKey="Lee, Su In" sort="Lee, Su In" uniqKey="Lee S" first="Su In" last="Lee">Su In Lee</name><name sortKey="Maharjan, Sony" sort="Maharjan, Sony" uniqKey="Maharjan S" first="Sony" last="Maharjan">Sony Maharjan</name><name sortKey="Park, Man Seong" sort="Park, Man Seong" uniqKey="Park M" first="Man-Seong" last="Park">Man-Seong Park</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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