Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning
Identifieur interne : 000A71 ( Pmc/Curation ); précédent : 000A70; suivant : 000A72Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning
Auteurs : Tiziano Gaiotto ; Simon E. HuftonSource :
- PLoS ONE [ 1932-6203 ] ; 2016.
Abstract
Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA) are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies) which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1)pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu), 2009 H1N1 pandemic (swine flu) and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our deep sequence analysis predicted that binding to the emerging H1N1 strain (A/Christchurch/16/2010) carrying the HA2-E47K mutation would not affect binding was confirmed experimentally. This demonstrates yeast display, in combination with deep sequencing, may be able to predict antibody reactivity to emerging influenza strains so assisting in the preparation for future influenza pandemics.
Url:
DOI: 10.1371/journal.pone.0164296
PubMed: 27741319
PubMed Central: 5065140
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning</title><author><name sortKey="Gaiotto, Tiziano" sort="Gaiotto, Tiziano" uniqKey="Gaiotto T" first="Tiziano" last="Gaiotto">Tiziano Gaiotto</name><affiliation><nlm:aff id="aff001"></nlm:aff></affiliation></author><author><name sortKey="Hufton, Simon E" sort="Hufton, Simon E" uniqKey="Hufton S" first="Simon E." last="Hufton">Simon E. Hufton</name><affiliation><nlm:aff id="aff001"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27741319</idno><idno type="pmc">5065140</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065140</idno><idno type="RBID">PMC:5065140</idno><idno type="doi">10.1371/journal.pone.0164296</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000A71</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A71</idno><idno type="wicri:Area/Pmc/Curation">000A71</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000A71</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning</title><author><name sortKey="Gaiotto, Tiziano" sort="Gaiotto, Tiziano" uniqKey="Gaiotto T" first="Tiziano" last="Gaiotto">Tiziano Gaiotto</name><affiliation><nlm:aff id="aff001"></nlm:aff></affiliation></author><author><name sortKey="Hufton, Simon E" sort="Hufton, Simon E" uniqKey="Hufton S" first="Simon E." last="Hufton">Simon E. Hufton</name><affiliation><nlm:aff id="aff001"></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA) are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies) which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1)pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu), 2009 H1N1 pandemic (swine flu) and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our deep sequence analysis predicted that binding to the emerging H1N1 strain (A/Christchurch/16/2010) carrying the HA2-E47K mutation would not affect binding was confirmed experimentally. This demonstrates yeast display, in combination with deep sequencing, may be able to predict antibody reactivity to emerging influenza strains so assisting in the preparation for future influenza pandemics.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Skehel, Jj" uniqKey="Skehel J">JJ Skehel</name></author><author><name sortKey="Wiley, Dc" uniqKey="Wiley D">DC Wiley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sriwilaijaroen, N" uniqKey="Sriwilaijaroen N">N Sriwilaijaroen</name></author><author><name sortKey="Suzuki, Y" uniqKey="Suzuki Y">Y Suzuki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Caton, Aj" uniqKey="Caton A">AJ Caton</name></author><author><name sortKey="Brownlee, Gg" uniqKey="Brownlee G">GG Brownlee</name></author><author><name sortKey="Yewdell, Jw" uniqKey="Yewdell J">JW Yewdell</name></author><author><name 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, CA USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27741319</article-id><article-id pub-id-type="pmc">5065140</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0164296</article-id><article-id pub-id-type="publisher-id">PONE-D-16-26003</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Physiology</subject><subj-group><subject>Immune Physiology</subject><subj-group><subject>Antibodies</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject><subj-group><subject>Physiology</subject><subj-group><subject>Immune Physiology</subject><subj-group><subject>Antibodies</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Immunology</subject><subj-group><subject>Immune System Proteins</subject><subj-group><subject>Antibodies</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject><subj-group><subject>Immunology</subject><subj-group><subject>Immune System Proteins</subject><subj-group><subject>Antibodies</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Proteins</subject><subj-group><subject>Immune System Proteins</subject><subj-group><subject>Antibodies</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Fungi</subject><subj-group><subject>Yeast</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Viruses</subject><subj-group><subject>RNA viruses</subject><subj-group><subject>Orthomyxoviruses</subject><subj-group><subject>Influenza viruses</subject><subj-group><subject>Influenza A virus</subject><subj-group><subject>H1N1</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Microbiology</subject><subj-group><subject>Medical microbiology</subject><subj-group><subject>Microbial pathogens</subject><subj-group><subject>Viral pathogens</subject><subj-group><subject>Orthomyxoviruses</subject><subj-group><subject>Influenza viruses</subject><subj-group><subject>Influenza A virus</subject><subj-group><subject>H1N1</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Medicine and health sciences</subject><subj-group><subject>Pathology and laboratory medicine</subject><subj-group><subject>Pathogens</subject><subj-group><subject>Microbial pathogens</subject><subj-group><subject>Viral pathogens</subject><subj-group><subject>Orthomyxoviruses</subject><subj-group><subject>Influenza viruses</subject><subj-group><subject>Influenza A virus</subject><subj-group><subject>H1N1</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Viruses</subject><subj-group><subject>Viral pathogens</subject><subj-group><subject>Orthomyxoviruses</subject><subj-group><subject>Influenza viruses</subject><subj-group><subject>Influenza A virus</subject><subj-group><subject>H1N1</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Genetics</subject><subj-group><subject>Mutation</subject><subj-group><subject>Point Mutation</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Spectrum Analysis Techniques</subject><subj-group><subject>Spectrophotometry</subject><subj-group><subject>Cytophotometry</subject><subj-group><subject>Flow Cytometry</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Chemical Characterization</subject><subj-group><subject>Binding Analysis</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Genetics</subject><subj-group><subject>Gene Identification and Analysis</subject><subj-group><subject>Mutation Detection</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Molecular Biology</subject><subj-group><subject>Molecular Biology Techniques</subject><subj-group><subject>Gene Mapping</subject><subj-group><subject>Epitope Mapping</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Molecular Biology Techniques</subject><subj-group><subject>Gene Mapping</subject><subj-group><subject>Epitope Mapping</subject></subj-group></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning</article-title><alt-title alt-title-type="running-head">Epitope Mapping of Nanobodies to Influenza Hemagglutinin</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Gaiotto</surname><given-names>Tiziano</given-names></name><xref ref-type="aff" rid="aff001"></xref></contrib><contrib contrib-type="author"><name><surname>Hufton</surname><given-names>Simon E.</given-names></name><xref ref-type="corresp" rid="cor001">*</xref><xref ref-type="aff" rid="aff001"></xref></contrib></contrib-group><aff id="aff001"><addr-line>Biotherapeutics Group, National Institute for Biological Standards and Control, a centre of the Medicines and Healthcare Products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG, United Kingdom</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Du</surname><given-names>Lanying</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>New York Blood Center, UNITED STATES</addr-line></aff><author-notes><fn fn-type="COI-statement" id="coi001"><p><bold>Competing Interests: </bold>An international patent application (PCT/GB2012/052164) covering the monoclonal antibodies described in this study has been filed. This does not alter adherence to all of the PLOS ONE policies on sharing data and information.</p></fn><fn fn-type="con"><p><list list-type="simple"><list-item><p><bold>Conceptualization:</bold> TG SEH.</p></list-item><list-item><p><bold>Investigation:</bold> TG SEH.</p></list-item><list-item><p><bold>Methodology:</bold> TG SEH.</p></list-item><list-item><p><bold>Project administration:</bold> SEH.</p></list-item><list-item><p><bold>Supervision:</bold> SEH.</p></list-item><list-item><p><bold>Visualization:</bold> TG.</p></list-item><list-item><p><bold>Writing – original draft:</bold> TG SEH.</p></list-item><list-item><p><bold>Writing – review & editing:</bold> TG SEH.</p></list-item></list></p></fn><corresp id="cor001">* E-mail: <email>simon.hufton@nibsc.org</email></corresp></author-notes><pub-date pub-type="epub"><day>14</day><month>10</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>11</volume><issue>10</issue><elocation-id>e0164296</elocation-id><history><date date-type="received"><day>29</day><month>6</month><year>2016</year></date><date date-type="accepted"><day>22</day><month>9</month><year>2016</year></date></history><permissions><copyright-statement>© 2016 Gaiotto, Hufton</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Gaiotto, Hufton</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="pone.0164296.pdf"></self-uri><abstract><p>Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA) are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies) which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1)pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu), 2009 H1N1 pandemic (swine flu) and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our deep sequence analysis predicted that binding to the emerging H1N1 strain (A/Christchurch/16/2010) carrying the HA2-E47K mutation would not affect binding was confirmed experimentally. This demonstrates yeast display, in combination with deep sequencing, may be able to predict antibody reactivity to emerging influenza strains so assisting in the preparation for future influenza pandemics.</p></abstract><funding-group><funding-statement>The authors have no funding or support to report.</funding-statement></funding-group><counts><fig-count count="7"></fig-count><table-count count="2"></table-count><page-count count="27"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its supporting information files. All sequencing datasets are available for download through accession number PRJEB15301 in ENA repository following link: <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ena/data/view/PRJEB15301">http://www.ebi.ac.uk/ena/data/view/PRJEB15301</ext-link>.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its supporting information files. All sequencing datasets are available for download through accession number PRJEB15301 in ENA repository following link: <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ena/data/view/PRJEB15301">http://www.ebi.ac.uk/ena/data/view/PRJEB15301</ext-link>.</p></notes></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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