Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein
Identifieur interne : 000740 ( Pmc/Corpus ); précédent : 000739; suivant : 000741Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein
Auteurs : Ann-Kathrin Haverkamp ; Berend J. Bosch ; Ingo Spitzbarth ; Annika Lehmbecker ; Nigeer Te ; Albert Bensaid ; Joaquim Segalés ; Wolfgang Baumg RtnerSource :
- Veterinary Immunology and Immunopathology [ 0165-2427 ] ; 2019.
Abstract
Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods.
Url:
DOI: 10.1016/j.vetimm.2019.109939
PubMed: 31526954
PubMed Central: 7112921
Links to Exploration step
PMC:7112921Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein</title><author><name sortKey="Haverkamp, Ann Kathrin" sort="Haverkamp, Ann Kathrin" uniqKey="Haverkamp A" first="Ann-Kathrin" last="Haverkamp">Ann-Kathrin Haverkamp</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Bosch, Berend J" sort="Bosch, Berend J" uniqKey="Bosch B" first="Berend J." last="Bosch">Berend J. Bosch</name><affiliation><nlm:aff id="aff0010">Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Spitzbarth, Ingo" sort="Spitzbarth, Ingo" uniqKey="Spitzbarth I" first="Ingo" last="Spitzbarth">Ingo Spitzbarth</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff0015">Center for Systems Neuroscience, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Lehmbecker, Annika" sort="Lehmbecker, Annika" uniqKey="Lehmbecker A" first="Annika" last="Lehmbecker">Annika Lehmbecker</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff0015">Center for Systems Neuroscience, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Te, Nigeer" sort="Te, Nigeer" uniqKey="Te N" first="Nigeer" last="Te">Nigeer Te</name><affiliation><nlm:aff id="aff0020">IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</nlm:aff></affiliation></author><author><name sortKey="Bensaid, Albert" sort="Bensaid, Albert" uniqKey="Bensaid A" first="Albert" last="Bensaid">Albert Bensaid</name><affiliation><nlm:aff id="aff0020">IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</nlm:aff></affiliation></author><author><name sortKey="Segales, Joaquim" sort="Segales, Joaquim" uniqKey="Segales J" first="Joaquim" last="Segalés">Joaquim Segalés</name><affiliation><nlm:aff id="aff0025">Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Barcelona, Spain</nlm:aff></affiliation><affiliation><nlm:aff id="aff0030">UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</nlm:aff></affiliation></author><author><name sortKey="Baumg Rtner, Wolfgang" sort="Baumg Rtner, Wolfgang" uniqKey="Baumg Rtner W" first="Wolfgang" last="Baumg Rtner">Wolfgang Baumg Rtner</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff0015">Center for Systems Neuroscience, 30559 Hannover, Germany</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31526954</idno><idno type="pmc">7112921</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112921</idno><idno type="RBID">PMC:7112921</idno><idno type="doi">10.1016/j.vetimm.2019.109939</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000740</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000740</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein</title><author><name sortKey="Haverkamp, Ann Kathrin" sort="Haverkamp, Ann Kathrin" uniqKey="Haverkamp A" first="Ann-Kathrin" last="Haverkamp">Ann-Kathrin Haverkamp</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Bosch, Berend J" sort="Bosch, Berend J" uniqKey="Bosch B" first="Berend J." last="Bosch">Berend J. Bosch</name><affiliation><nlm:aff id="aff0010">Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, the Netherlands</nlm:aff></affiliation></author><author><name sortKey="Spitzbarth, Ingo" sort="Spitzbarth, Ingo" uniqKey="Spitzbarth I" first="Ingo" last="Spitzbarth">Ingo Spitzbarth</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff0015">Center for Systems Neuroscience, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Lehmbecker, Annika" sort="Lehmbecker, Annika" uniqKey="Lehmbecker A" first="Annika" last="Lehmbecker">Annika Lehmbecker</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff0015">Center for Systems Neuroscience, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Te, Nigeer" sort="Te, Nigeer" uniqKey="Te N" first="Nigeer" last="Te">Nigeer Te</name><affiliation><nlm:aff id="aff0020">IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</nlm:aff></affiliation></author><author><name sortKey="Bensaid, Albert" sort="Bensaid, Albert" uniqKey="Bensaid A" first="Albert" last="Bensaid">Albert Bensaid</name><affiliation><nlm:aff id="aff0020">IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</nlm:aff></affiliation></author><author><name sortKey="Segales, Joaquim" sort="Segales, Joaquim" uniqKey="Segales J" first="Joaquim" last="Segalés">Joaquim Segalés</name><affiliation><nlm:aff id="aff0025">Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Barcelona, Spain</nlm:aff></affiliation><affiliation><nlm:aff id="aff0030">UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</nlm:aff></affiliation></author><author><name sortKey="Baumg Rtner, Wolfgang" sort="Baumg Rtner, Wolfgang" uniqKey="Baumg Rtner W" first="Wolfgang" last="Baumg Rtner">Wolfgang Baumg Rtner</name><affiliation><nlm:aff id="aff0005">Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff0015">Center for Systems Neuroscience, 30559 Hannover, Germany</nlm:aff></affiliation></author></analytic><series><title level="j">Veterinary Immunology and Immunopathology</title><idno type="ISSN">0165-2427</idno><idno type="eISSN">1873-2534</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 (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Adney, D R" uniqKey="Adney D">D.R. Adney</name></author><author><name sortKey="Bielefeldt Ohmann, H" uniqKey="Bielefeldt Ohmann H">H. Bielefeldt-Ohmann</name></author><author><name sortKey="Hartwig, A E" uniqKey="Hartwig A">A.E. Hartwig</name></author><author><name sortKey="Bowen, R A" uniqKey="Bowen R">R.A. Bowen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Adney, D R" uniqKey="Adney D">D.R. Adney</name></author><author><name sortKey="Brown, V R" uniqKey="Brown V">V.R. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Vet Immunol Immunopathol</journal-id><journal-id journal-id-type="iso-abbrev">Vet. Immunol. Immunopathol</journal-id><journal-title-group><journal-title>Veterinary Immunology and Immunopathology</journal-title></journal-title-group><issn pub-type="ppub">0165-2427</issn><issn pub-type="epub">1873-2534</issn><publisher><publisher-name>Elsevier B.V.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31526954</article-id><article-id pub-id-type="pmc">7112921</article-id><article-id pub-id-type="publisher-id">S0165-2427(19)30171-0</article-id><article-id pub-id-type="doi">10.1016/j.vetimm.2019.109939</article-id><article-id pub-id-type="publisher-id">109939</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein</article-title></title-group><contrib-group><contrib contrib-type="author" id="aut0005"><name><surname>Haverkamp</surname><given-names>Ann-Kathrin</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="fn0005" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="aut0010"><name><surname>Bosch</surname><given-names>Berend J.</given-names></name><xref rid="aff0010" ref-type="aff">b</xref><xref rid="fn0005" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="aut0015"><name><surname>Spitzbarth</surname><given-names>Ingo</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="aff0015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="aut0020"><name><surname>Lehmbecker</surname><given-names>Annika</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="aff0015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="aut0025"><name><surname>Te</surname><given-names>Nigeer</given-names></name><xref rid="aff0020" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="aut0030"><name><surname>Bensaid</surname><given-names>Albert</given-names></name><xref rid="aff0020" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="aut0035"><name><surname>Segalés</surname><given-names>Joaquim</given-names></name><xref rid="aff0025" ref-type="aff">e</xref><xref rid="aff0030" ref-type="aff">f</xref></contrib><contrib contrib-type="author" id="aut0040"><name><surname>Baumgärtner</surname><given-names>Wolfgang</given-names></name><email>Wolfgang.Baumgaertner@tiho-hannover.de</email><xref rid="aff0005" ref-type="aff">a</xref><xref rid="aff0015" ref-type="aff">c</xref><xref rid="cor0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0005"><label>a</label>Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany</aff><aff id="aff0010"><label>b</label>Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, the Netherlands</aff><aff id="aff0015"><label>c</label>Center for Systems Neuroscience, 30559 Hannover, Germany</aff><aff id="aff0020"><label>d</label>IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</aff><aff id="aff0025"><label>e</label>Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Barcelona, Spain</aff><aff id="aff0030"><label>f</label>UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain</aff><author-notes><corresp id="cor0005"><label>⁎</label>Corresponding author at: Department of Pathology, University of Veterinary Medicine Hannover Foundation, Bünteweg 17, 30559 Hannover, Germany. <email>Wolfgang.Baumgaertner@tiho-hannover.de</email></corresp><fn id="fn0005"><label>1</label><p id="npar0005">both authors contributed equally.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>9</day><month>9</month><year>2019</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>12</month><year>2019</year></pub-date><pub-date pub-type="epub"><day>9</day><month>9</month><year>2019</year></pub-date><volume>218</volume><fpage>109939</fpage><lpage>109939</lpage><history><date date-type="received"><day>10</day><month>5</month><year>2019</year></date><date date-type="rev-recd"><day>2</day><month>9</month><year>2019</year></date><date date-type="accepted"><day>8</day><month>9</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 Elsevier B.V. All rights reserved.</copyright-statement><copyright-year>2019</copyright-year><copyright-holder>Elsevier B.V.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract id="abs0005"><p>Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods.</p></abstract><kwd-group id="kwd0005"><title>Keywords</title><kwd>Immunohistochemistry</kwd><kwd>Middle East respiratory syndrome coronavirus</kwd><kwd>Spike protein</kwd><kwd>Monoclonal human antibodies</kwd></kwd-group></article-meta></front><body><sec id="sec0005"><label>1</label><title>Introduction</title><p id="par0005">Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel lineage C betacoronavirus, which was first identified in a patient from the Kingdom of Saudi Arabia in June 2012 (<xref rid="bib0205" ref-type="bibr">Zaki et al., 2012</xref>). As of May 2019, MERS-CoV has been detected in 27 countries and infected more than 2300 patients (World Health Organization (2019) Middle East respiratory syndrome coronavirus, available at <ext-link ext-link-type="uri" xlink:href="https://www.who.int/csr/don/24-April-2019-mers-saudi-arabia/en/" id="intr0005">https://www.who.int/csr/don/24-April-2019-mers-saudi-arabia/en/</ext-link>). Although the disease vanished almost completely from headlines and newspapers, it is still a potential threat to human health since approximately one third of all affected patients succumb to acute respiratory distress syndrome and renal dysfunction (<xref rid="bib0210" ref-type="bibr">Zumla et al., 2015</xref>).</p><p id="par0010">Even though MERS-CoV is most likely derived from an ancestral reservoir in bats (<xref rid="bib0025" ref-type="bibr">Corman et al., 2014</xref>; <xref rid="bib0035" ref-type="bibr">Cui et al., 2013</xref>; <xref rid="bib0045" ref-type="bibr">de Wit and Munster, 2013</xref>; <xref rid="bib0115" ref-type="bibr">Memish et al., 2013</xref>; <xref rid="bib0125" ref-type="bibr">Munster et al., 2016</xref>; <xref rid="bib0180" ref-type="bibr">van Boheemen et al., 2012</xref>), transmission to humans occurs mainly <italic>via</italic> dromedaries, which represent a major reservoir for the virus in large parts of the Middle East, the Canary Islands, Africa, and Southern Asia (<xref rid="bib0060" ref-type="bibr">Falzarano et al., 2017</xref>; <xref rid="bib0065" ref-type="bibr">Haagmans et al., 2014</xref>; <xref rid="bib0080" ref-type="bibr">Hemida et al., 2017</xref>; <xref rid="bib0160" ref-type="bibr">Saqib et al., 2017</xref>). Infection has also been demonstrated in farmed alpacas from a region of Qatar where MERS-CoV is endemic (<xref rid="bib0155" ref-type="bibr">Reusken et al., 2016</xref>), but not in any other livestock species (<xref rid="bib0150" ref-type="bibr">Reusken et al., 2013</xref>). Recently, experimental inoculation of pigs, alpacas, and llamas with MERS-CoV resulted in low levels of viral replication in pigs and moderate to high levels of viral replication and shedding in the latter species, respectively (<xref rid="bib0005" ref-type="bibr">Adney et al., 2016a</xref>; <xref rid="bib0040" ref-type="bibr">de Wit et al., 2017</xref>; <xref rid="bib0185" ref-type="bibr">Vergara-Alert et al., 2017</xref>). Similarly, minimal virus shedding was detected in goats, sheep, and horses after experimental infection, but only goats developed neutralizing antibodies (<xref rid="bib0010" ref-type="bibr">Adney et al., 2016b</xref>). Due to the close interactions of humans with potentially susceptible and contagious livestock species in certain parts of the world, the development of an effective vaccination strategy against MERS-CoV was proposed early after its initial discovery and was recently followed up by the development of protective antibodies by different protocols (<xref rid="bib0020" ref-type="bibr">Agrawal et al., 2016</xref>; <xref rid="bib0030" ref-type="bibr">Corti et al., 2015</xref>; <xref rid="bib0085" ref-type="bibr">Houser et al., 2016</xref>; <xref rid="bib0090" ref-type="bibr">Jiang et al., 2014</xref>; <xref rid="bib0095" ref-type="bibr">Johnson et al., 2016</xref>; <xref rid="bib0105" ref-type="bibr">Li et al., 2015</xref>; <xref rid="bib0135" ref-type="bibr">Pascal et al., 2015</xref>; <xref rid="bib0140" ref-type="bibr">Qiu et al., 2016</xref>; <xref rid="bib0175" ref-type="bibr">Tang et al., 2014</xref>; <xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). Widjaja and colleagues (2019) developed a set of protective neutralizing and non-neutralizing human monoclonal antibodies (mAbs) which target different epitopes of the MERS-CoV spike (MERS-S) protein. The MERS-S protein is known to represent a key target for the development of new therapeutics and consists of a receptor-binding subunit S1 and a membrane-fusion subunit S2 (<xref rid="bib0050" ref-type="bibr">Du et al., 2017</xref>). The subunit S1 is, in turn, composed of four different core domains. The domain S1B binds to the host-cell receptor dipeptidylpeptidase 4 (DPP4; <xref rid="bib0110" ref-type="bibr">Lu et al., 2013</xref>; <xref rid="bib0145" ref-type="bibr">Raj et al., 2013</xref>; <xref rid="bib0195" ref-type="bibr">Wang et al., 2013</xref>), while the domain S1A binds to sialoglycans which enhances infection of human lung cells by MERS-CoV (<xref rid="bib0100" ref-type="bibr">Li et al., 2017</xref>). The subunit S2 comprises the fusion peptide, two heptad repeats and a transmembrane domain, which mediate fusion of the virus with the cell membrane (<xref rid="bib0110" ref-type="bibr">Lu et al., 2013</xref>; <xref rid="bib0130" ref-type="bibr">Pallesen et al., 2017</xref>; <xref rid="bib0145" ref-type="bibr">Raj et al., 2013</xref>; <xref rid="bib0195" ref-type="bibr">Wang et al., 2013</xref>).</p><p id="par0015">In the present study, these mAbs were tested for immunohistochemistry to detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV infected alpacas. Immunohistochemistry is a well-established and powerful method which is frequently used for diagnostic purposes and evaluation of animal experiments. Simple protocols allow precise correlation between the presence and severity of lesions and the amount of viral antigen in the respective localization (<xref rid="bib0055" ref-type="bibr">Duraiyan et al., 2012</xref>). Even though there are several mono- and polyclonal antibodies available for the detection of the MERS-CoV nucleocapsid and spike protein on FFPE tissue (<xref rid="bib0075" ref-type="bibr">Haverkamp et al., 2018</xref>), these antibodies are all derived from rabbits and mice, respectively (<ext-link ext-link-type="uri" xlink:href="https://www.biocompare.com" id="intr0010">https://www.biocompare.com</ext-link>, accessed March 11, 2019). However, for double or triple immunohistochemistry and -fluoresence labeling, it can be useful to have access to epitope-specific antibodies from different species, which can be used to visualize viral antigen in tissue sections. It was therefore the aim of the present study to evaluate the suitability of different human anti-MERS-S mAbs for detection of the respective antigen on FFPE material.</p></sec><sec id="sec0010"><label>2</label><title>Materials and methods</title><sec id="sec0015"><label>2.1</label><title>Human monoclonal antibodies</title><p id="par0020">The development of the eight diverse human mAbs directed against different epitopes of MERS-S protein has been published previously (<xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). Shortly, generation of anti-MERS-S H2L2 mAbs was achieved by recurrent immunization of transgenic H2L2 mice, which encode the human immunoglobulin variable regions, with the respective antigens and adjuvants (for preparation of antigens see original publications (<xref rid="bib0100" ref-type="bibr">Li et al., 2017</xref>; <xref rid="bib0120" ref-type="bibr">Mou et al., 2013</xref>; <xref rid="bib0190" ref-type="bibr">Walls et al., 2016</xref>; <xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>)). At day 74, four days after the last immunization, spleen and lymph nodes were harvested and hybridoma cell lines were generated using the SP2/0 myeloma cell line. For production of recombinant mAbs, cDNA’s encoding the variable heavy and light chain regions of anti-MERS-S H2L2 mAbs were cloned into expression plasmids containing the human IgG1 heavy chain and Ig kappa light chain constant regions, respectively. Recombinant human anti-MERS-S mAbs were produced in HEK-293 T cells following co-transfection of the IgG1 heavy and light chain expression plasmids (Invivogen) according to manufacturers’ protocols (<xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). Antibodies were purified from tissue culture supernatants using Protein-A affinity chromatography. Purified antibodies were quantified using UV absorbance (280 nm) and Coomassie stained SDS-PAGE analysis was applied in addition. Antibodies were stored at 4 °C until further use. After screening for MERS-S reactivity and neutralizing capacity eight different mAbs (<xref rid="tbl0005" ref-type="table">Table 1</xref>) were selected based on epitope distribution and neutralization.<table-wrap position="float" id="tbl0005"><label>Table 1</label><caption><p>Antibodies, clonality, species, antigen retrieval, dilution, secondary antibodies and results.</p></caption><alt-text id="at0020">Table 1</alt-text><table frame="hsides" rules="groups"><thead><tr><th align="left">Antibody</th><th align="left">Clonality, host species (lot number)</th><th align="left">Protein content [μg/ml]</th><th align="left">Antigen retrieval</th><th align="left">Dilution</th><th align="left">Secondary antibody</th><th align="left">Result</th></tr></thead><tbody><tr><td align="left">1.2g5</td><td align="left" valign="middle" rowspan="8">mc, human</td><td align="left">0.23</td><td align="left">#</td><td align="left">1:5, 1:10, 1:20</td><td align="left" valign="middle" rowspan="8">GaH-b</td><td align="left">+</td></tr><tr><td align="left">1.10f3</td><td align="left">0.27</td><td align="left">#</td><td align="left">1:5, 1:10, 1:20</td><td align="left">(+)</td></tr><tr><td align="left">1.6c7</td><td align="left">0.27</td><td align="left" valign="middle" rowspan="8">citrate</td><td align="left">1:5</td><td align="left">(+)</td></tr><tr><td align="left">1.6f9</td><td align="left">0.23</td><td align="left">1:5</td><td align="left">–</td></tr><tr><td align="left">4.6e10</td><td align="left">0.43</td><td align="left">1:5</td><td align="left">–</td></tr><tr><td align="left">7.7g6</td><td align="left">0.77</td><td align="left">1:5</td><td align="left">–</td></tr><tr><td align="left">1.8e5</td><td align="left">0.25</td><td align="left">1:5</td><td align="left">–</td></tr><tr><td align="left">3.5g6</td><td align="left">0.91</td><td align="left">1:5</td><td align="left">–</td></tr><tr><td align="left">MERS-CoV nucleocapsid (Sino Biological)</td><td align="left">mc, mouse (HB10AP1804-B)</td><td align="left">1.0</td><td align="left">1:70</td><td align="left">GaM-b</td><td align="left">+++</td></tr><tr><td align="left">MERS-CoV nucleocapsid (Sino Biological)</td><td align="left">pc, rabbit (HB07AP1208)</td><td align="left">1.0</td><td align="left">1:2000</td><td align="left">GaR-b</td><td align="left">+++</td></tr></tbody></table><table-wrap-foot><fn><p>GaH-b, goat anti-human IgG biotinylated; GaM-b, goat anti-mouse IgG biotinylated; GaR-b, goat anti-rabbit IgG biotinylated; mc, monoclonal; MERS-CoV, Middle East respiratory syndrome coronavirus; pc, polyclonal; #, antigen retrieval was performed with boiling citrate buffer, proteinase K and omission of pretreatment; +++, intense antigen specific signal in several cells, minimal background staining (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>C, 1D); +, moderately intense antigen specific signal in fewer cells, minimal background staining (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>A); (+), moderately intense antigen specific signal in fewer cells, strong background staining (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>C, D); -, no antigen specific signal, varying degrees of background staining.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="sec0020"><label>2.2</label><title>Tissue</title><p id="par0025">Formalin-fixed tissue of nasal turbinates from two experimentally infected alpacas was obtained from an animal trial recently implemented at the Research Center for Animal Health (<italic>Centre de Recerca en Sanitat Animal</italic>, CReSA, Barcelona, Spain). The mucosa was carefully removed from underlying bone and embedded in paraffin according to a routinely used protocol. Serial sections of 4 μm thickness were mounted on coated glass slides (Superfrost Plus®, Menzel Co.) and the first section was stained with HE for evaluation by light microscopy and subsequent sections were processed for immunohistochemistry. For negative control, nasal turbinate of a non-infected alpaca (collected during routine necropsy at the Department of Pathology, University of Veterinary Medicine Hannover, Germany) was used. The animal died due to cachexia related to non-respiratory disease.</p></sec><sec id="sec0025"><label>2.3</label><title>Immunohistochemistry</title><p id="par0030">Sections were dewaxed in Roticlear® (Roth C. GmbH & Co. KG) and subsequently rehydrated in isopropanol and 96% ethanol (Roth C. GmbH & Co. KG). Endogenous peroxidase activity was blocked by incubation of sections in 85% ethanol with 0.5% H<sub>2</sub>O<sub>2</sub> (VWRTM International GmbH) for 30 min at room temperature and antigen retrieval was performed by incubation in citrate buffer (2.1 g citric acid monohydrate in 1 l distilled water, adjusted with NaOH to pH = 6.0) for 20 min in a microwave (800 W). In a preliminary experiment two of the eight antibodies (1.10f3 and 1.2g5) were additionally tested without pretreatment and with enzymatic digestion by Proteinase K antigen retrieval solution (Merck KGaA) according to the manufacturer’s protocol. All sections were transferred to Shandon Coverplates™ (Thermo Electron GmbH) and nonspecific binding was blocked by inactivated 20% goat serum diluted in phosphate buffered saline (PBS) including 1% bovine serum albumin (BSA; PBS/BSA) for 30 min. Afterwards sections were incubated with the primary antibody (1.2g5, 1.6c7, 1.10f3, 1.6f9, 4.6e10, 7.7g6, 3.5g6, and 1.8e5) in their respective dilution (<xref rid="tbl0005" ref-type="table">Table 1</xref>) for 90 min at room temperature and the secondary biotinylated antibody diluted in PBS (1:200) was added. Incubation for 60 min at room temperature was followed by treatment with the avidin-biotin-peroxidase complex (Vectastain ABC Kit Standard, VectorLaboratories) according to the manufacturer’s protocol. Visualization of the reaction was achieved by addition of chromogen 3,3-diaminobenzidine tetrahydrochloride (DAB, 0.05%, Sigma Aldrich Chemie GmbH) and 0.03% H<sub>2</sub>O<sub>2</sub>. Sections were finally slightly counterstained with Mayer’s hematoxylin (Roth C. GmbH & Co KG). For positive control and elucidation of virus distribution, additional sections were stained with two different commercially available, previously published monoclonal mouse and polyclonal rabbit anti-MERS-CoV nucleocapsid antibodies (Sino Biological Inc.; <xref rid="bib0070" ref-type="bibr">Haagmans et al., 2016</xref>; <xref rid="bib0075" ref-type="bibr">Haverkamp et al., 2018</xref>), respectively. For negative controls, the primary antibody was replaced by ascites fluid from Balb/c mice (1:1,000) and normal rabbit serum (1:3,000), respectively. Moreover, all antibodies were applied to tissue of a non-infected alpaca (section <xref rid="sec0020" ref-type="sec">2.2</xref>).</p></sec></sec><sec id="sec0030"><label>3</label><title>Results and discussion</title><p id="par0035">Evaluation of HE stained slides by light microscopy revealed a mild to moderate, multifocal, lymphoplasmahistiocytic and suppurative rhinitis characterized by low numbers of intraepithelial neutrophils and lymphocytes and infiltration of the lamina propria and submucosa by low to moderate numbers of lymphocytes, single plasma cells, and macrophages (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>A, B). These findings are reminiscent of previous observations in other large animal species though lesions in alpacas are relatively mild and lack the necrotizing component that has been described for dromedaries, llamas, and pigs (<xref rid="bib0015" ref-type="bibr">Adney et al., 2014</xref>; <xref rid="bib0070" ref-type="bibr">Haagmans et al., 2016</xref>; <xref rid="bib0185" ref-type="bibr">Vergara-Alert et al., 2017</xref>). The commercially available monoclonal and polyclonal anti-MERS-CoV nucleocapsid antibodies, which were used as positive controls, revealed a strong intracytoplasmic and cilia-associated staining pattern in numerous cells (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>C, D) according to formerly published results (<xref rid="bib0070" ref-type="bibr">Haagmans et al., 2016</xref>; <xref rid="bib0075" ref-type="bibr">Haverkamp et al., 2018</xref>). A specific staining was absent in all negative controls as expected.<fig id="fig0005"><label>Fig. 1</label><caption><p>Representative HE findings in respiratory epithelium of the nasal turbinates of MERS-CoV-infected alpacas and immunohistochemical reactions of the commercially available positive controls for comparison. (A) Multifocal infiltration of lamina propria and submucosa by moderate numbers of lymphocytes, macrophages, and single neutrophilic granulocytes (asterisk). (B) Exocytosis of single neutrophilic granulocytes (grey arrow). (C, D) Abundant viral antigen was detected multifocally in the cytoplasm and along the apical membranous region of epithelial cells using a monoclonal mouse (C, Sino Biological Inc.) and polyclonal rabbit anti-MERS-CoV nucleocapsid antibody (D, Sino Biological Inc.) with citrate pretreatment, in a dilution of 1:70 and 1:2,000, respectively. Both antibodies exhibited a similar staining intensity. (A, B) HE staining; 400x, (C, D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.</p></caption><alt-text id="at0005">Fig. 1</alt-text><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="par0040">For evaluation of human mAbs by immunohistochemistry, two of the tested antibodies (1.2g5 and 1.10f3) were applied in three different dilutions and three different pretreatments in a preliminary experiment to identify the best method for the use of human mAbs on FFPE tissue (<xref rid="tbl0005" ref-type="table">Table 1</xref>). As a result, heat-induced antigen retrieval and application of the antibody in the lowest dilution revealed the best staining effects. According to these results, all other antibodies (1.6c7, 1.6f9, 4.6e10, 7.7g6, 3.5g6, 1.8e5) were tested with heat-induced antigen retrieval and in the lowest dilution.</p><p id="par0045">The use of the 1.2g5 antibody in a 1:5 dilution with heat-induced antigen retrieval showed the strongest signal of all tested non-commercially available antibodies. Detected antigen was present multifocally in the cytoplasm of ciliated respiratory epithelium and segmentally along the apical membranous region of few infected cells (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>A). The observed staining pattern was comparable to descriptions by others investigating the distribution of MERS-CoV in tissue sections of different infected animal species (<xref rid="bib0015" ref-type="bibr">Adney et al., 2014</xref>; <xref rid="bib0070" ref-type="bibr">Haagmans et al., 2016</xref>; <xref rid="bib0075" ref-type="bibr">Haverkamp et al., 2018</xref>; <xref rid="bib0185" ref-type="bibr">Vergara-Alert et al., 2017</xref>) but the number of labeled cells was considerably lower. Staining with the same antibody without pretreatment revealed a similar distribution of viral antigen in a comparable number of cells but a less prominent expression pattern (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>B), which is most likely linked to the lack of antigen unmasking which takes place during antigen retrieval (<xref rid="bib0165" ref-type="bibr">Shi et al., 2011</xref>). Detection of MERS-CoV antigen by the 1.2g5 antibody was almost completely absent with pretreatment by enzymatic digestion in all three dilutions, resulting only in a pale brownish signal interpreted as non-specific background staining (data not shown). The antibody 1.10f3 showed a strong non-specific background staining in all dilutions and pretreatments. A moderately intense antigen-specific signal was detected multifocally in cilia of few cells using the lowest dilution and citrate pretreatment (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>C).<fig id="fig0010"><label>Fig. 2</label><caption><p>Representative positive immunohistochemical reactions for human monoclonal antibodies. (A) Antibody 1.2g5 with citrate pretreatment exhibited a strong multifocal MERS-CoV antigen specific signal in the cytoplasm of ciliated respiratory epithelial cells (arrow) and segmentally along the apical membranous region (arrow heads). (B) The same protocol without pretreatment revealed a much fainter but similarly distributed staining of cytoplasm (arrow) and ciliary base (arrow heads). (C) Antibody 1.10f3 with citrate pretreatment displayed a strong diffuse background staining and only few positively stained cilia (arrowhead). Single intraepithelial plasma cells displayed a strong false positive intracytoplasmic staining (white arrow). (D) Antibody 1.6c7 with citrate pretreatment exhibited also a strong diffuse background staining and only few positive staining cilia (arrow heads). (A–D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.</p></caption><alt-text id="at0010">Fig. 2</alt-text><graphic xlink:href="gr2_lrg"></graphic></fig></p><p id="par0050">Of the six remaining antibodies (1.6c7, 1.6f9, 4.6e10, 7.7g6, 3.5g6, 1.8e5), only antibody 1.6c7 showed a mild multifocal staining of cilia accompanied by a strong background reactivity (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>D). The other five antibodies (1.6f9, 4.6e10, 7.7g6, 1.8e5, 3.5g6) did not reveal any recognizable specific staining of MERS-CoV antigen on FFPE tissue along with no to very mild (1.6f9, 4.6e10, <xref rid="fig0015" ref-type="fig">Fig. 3</xref>A, B), mild (7.7g6, 1.8e5, <xref rid="fig0015" ref-type="fig">Fig. 3</xref>C, D) and strong (3.5g6, <xref rid="fig0015" ref-type="fig">Fig. 3</xref>E) non-specific background staining. By contrast, all antibodies displayed a distinct signal by immunofluorescence staining of MERS-CoV-infected Huh-7 cells in culture (<xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). Possible explanations for the lack of immunoreactivity on FFPE tissue include effacement of conformational epitopes by formalin fixation and/or antigen retrieval protocols (<xref rid="bib0170" ref-type="bibr">Smedley et al., 2007</xref>).<fig id="fig0015"><label>Fig. 3</label><caption><p>Representative negative immunohistochemical reactions for human monoclonal antibodies. (A–E) Specific staining for MERS-CoV antigen was absent for the antibodies 1.6f9 (A), 4.6e10 (B), 7.7g6 (C), 1.8e5 (D), and 3.5g6 (E), respectively. Reactions were accompanied by mild to severe non-specific, intracytoplasmic background staining. (A–E) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.</p></caption><alt-text id="at0015">Fig. 3</alt-text><graphic xlink:href="gr3_lrg"></graphic></fig></p><p id="par0055">The tested antibodies in the present study were derived from transgenic mice expressing chimeric antibodies containing human variable domains and constant antibody domains of rat origin. For generation of fully human antibodies the human variable heavy and light chain encoding gene segments of the antibodies isolated from MERS-S immunized mice transgenic for human V genes were cloned into human IgG1 heavy and light chain expression vectors, respectively (<xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). All these antibodies are directed against MERS-S and belong to six distinct epitope groups that interfere with three critical entry functions <italic>in vivo</italic>. In particular, 1.2g5, 7.7g6, 1.6f9, 1.8e5, and 4.6e10 are neutralizing antibodies directed against the receptor binding domain (RBD) S1B of the MERS-S protein (<xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>), which facilitates virus binding to the DPP4 receptor on the host cell surface (<xref rid="bib0120" ref-type="bibr">Mou et al., 2013</xref>; <xref rid="bib0145" ref-type="bibr">Raj et al., 2013</xref>). The neutralizing antibodies 1.6c7 and 3.5g6 bind to the ectodomain of the membrane fusion subunit S2 which mediates fusion of the viral and cellular membranes (<xref rid="bib0100" ref-type="bibr">Li et al., 2017</xref>; <xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). By contrast, antibody 1.10f3 is a non-neutralizing antibody that recognizes the sialic acid binding domain (<xref rid="bib0200" ref-type="bibr">Widjaja et al., 2019</xref>). Since the positive staining antibodies 1.2g5, 1.10f3 and 1.6c7 recognize epitopes that belong to different functional groups (DPP4 binding, sialic acid binding and membrane fusion, respectively), the suitability of mAbs for use in immunohistochemistry cannot be predicted from the function of their target epitopes <italic>in vivo</italic>.</p></sec><sec id="sec0035"><label>4</label><title>Conclusion</title><p id="par0060">The present study shows that three tested human mAbs, designated 1.2g5, 1.10f3, and 1.6c7, are suitable to detect MERS-CoV antigen on FFPE tissue sections of experimentally infected alpacas, though to a limited extend. The best immunolabelling results for all three antibodies were achieved at the lowest dilution (1:5) with heat-induced antigen retrieval. However, results demonstrate that the suitability of the antibodies was unrelated to the function of their target epitopes <italic>in vivo</italic> and that commercially available monoclonal and polyclonal anti-nucleocapsid antibodies generally appear more suitable to detect MERS-CoV antigen on FFPE sections in routine diagnostics. 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Med.</source><volume>367</volume><year>2012</year><fpage>1814</fpage><lpage>1820</lpage><pub-id pub-id-type="pmid">23075143</pub-id></element-citation></ref><ref id="bib0210"><element-citation publication-type="journal" id="sbref0210"><person-group person-group-type="author"><name><surname>Zumla</surname><given-names>A.</given-names></name><name><surname>Hui</surname><given-names>D.S.</given-names></name><name><surname>Perlman</surname><given-names>S.</given-names></name></person-group><article-title>Middle east respiratory syndrome</article-title><source>Lancet</source><volume>386</volume><year>2015</year><fpage>995</fpage><lpage>1007</lpage><pub-id pub-id-type="pmid">26049252</pub-id></element-citation></ref></ref-list><ack id="ack0005"><title>Acknowledgements</title><p>The authors would like to thank Petra Grünig, Caroline Schütz, David Solanes, Xavier Abad, and Ivan Cordón for excellent technical assistance. This research was performed as part of the Zoonoses Anticipation and Preparedness Initiative (ZAPI project; IMI Grant Agreement no. 115760), with the assistance and financial support of <funding-source id="gs0005">IMI</funding-source> and the <funding-source id="gs0010">European Commission</funding-source>, and in-kind contributions from <funding-source id="gs0015">EFPIA partners</funding-source>. The animal experiments at CReSA, IRTA-UAB were supported by the <funding-source id="gs0020">VetBioNet project</funding-source> (EU Grant Agreement INFRA-2016-1 Nº731014).</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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