Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines
Identifieur interne : 000080 ( Pmc/Checkpoint ); précédent : 000079; suivant : 000081Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines
Auteurs : Laura Rodriguez ; Pilar Blanco-Lobo ; Emma C. Reilly [États-Unis] ; Tatsuya Maehigashi ; Aitor Nogales ; Andrew Smith [États-Unis] ; David J. Topham [États-Unis] ; Stephen Dewhurst ; Baek Kim [Corée du Sud] ; Luis Martínez-SobridoSource :
- Viruses [ 1999-4915 ] ; 2019.
Abstract
Influenza viruses cause annual, seasonal infection across the globe. Vaccination represents the most effective strategy to prevent such infections and/or to reduce viral disease. Two major types of influenza vaccines are approved for human use: inactivated influenza vaccines (IIVs) and live attenuated influenza vaccines (LAIVs). Two Master Donor Virus (MDV) backbones have been used to create LAIVs against influenza A virus (IAV): the United States (US) A/Ann Arbor/6/60 (AA) and the Russian A/Leningrad/134/17/57 (Len) H2N2 viruses. The mutations responsible for the temperature sensitive (
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DOI: 10.3390/v11100928
PubMed: 31658679
PubMed Central: 6832241
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines</title><author><name sortKey="Rodriguez, Laura" sort="Rodriguez, Laura" uniqKey="Rodriguez L" first="Laura" last="Rodriguez">Laura Rodriguez</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Blanco Lobo, Pilar" sort="Blanco Lobo, Pilar" uniqKey="Blanco Lobo P" first="Pilar" last="Blanco-Lobo">Pilar Blanco-Lobo</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Reilly, Emma C" sort="Reilly, Emma C" uniqKey="Reilly E" first="Emma C." last="Reilly">Emma C. Reilly</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="af2-viruses-11-00928">David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Maehigashi, Tatsuya" sort="Maehigashi, Tatsuya" uniqKey="Maehigashi T" first="Tatsuya" last="Maehigashi">Tatsuya Maehigashi</name><affiliation><nlm:aff id="af3-viruses-11-00928">Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia, GA 30322, USA;<email>t.maehigashi@emory.edu</email> (T.M.);<email>baek.kim@emory.edu</email> (B.K.)</nlm:aff></affiliation></author><author><name sortKey="Nogales, Aitor" sort="Nogales, Aitor" uniqKey="Nogales A" first="Aitor" last="Nogales">Aitor Nogales</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Smith, Andrew" sort="Smith, Andrew" uniqKey="Smith A" first="Andrew" last="Smith">Andrew Smith</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="af4-viruses-11-00928">Medical Scientist Training Program (MSTP), University of Rochester, Rochester, New York, NY 14642, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Medical Scientist Training Program (MSTP), University of Rochester, Rochester, New York, NY 14642</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Topham, David J" sort="Topham, David J" uniqKey="Topham D" first="David J." last="Topham">David J. Topham</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="af2-viruses-11-00928">David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Dewhurst, Stephen" sort="Dewhurst, Stephen" uniqKey="Dewhurst S" first="Stephen" last="Dewhurst">Stephen Dewhurst</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Kim, Baek" sort="Kim, Baek" uniqKey="Kim B" first="Baek" last="Kim">Baek Kim</name><affiliation><nlm:aff id="af3-viruses-11-00928">Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia, GA 30322, USA;<email>t.maehigashi@emory.edu</email> (T.M.);<email>baek.kim@emory.edu</email> (B.K.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af5-viruses-11-00928">College of Pharmacy, Kyung-Hee University, Seoul 02447, Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>College of Pharmacy, Kyung-Hee University, Seoul 02447</wicri:regionArea><placeName><settlement type="city">Séoul</settlement><region type="capital">Région capitale de Séoul</region></placeName></affiliation></author><author><name sortKey="Martinez Sobrido, Luis" sort="Martinez Sobrido, Luis" uniqKey="Martinez Sobrido L" first="Luis" last="Martínez-Sobrido">Luis Martínez-Sobrido</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31658679</idno><idno type="pmc">6832241</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832241</idno><idno type="RBID">PMC:6832241</idno><idno type="doi">10.3390/v11100928</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000C50</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000C50</idno><idno type="wicri:Area/Pmc/Curation">000C50</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000C50</idno><idno type="wicri:Area/Pmc/Checkpoint">000080</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000080</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines</title><author><name sortKey="Rodriguez, Laura" sort="Rodriguez, Laura" uniqKey="Rodriguez L" first="Laura" last="Rodriguez">Laura Rodriguez</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Blanco Lobo, Pilar" sort="Blanco Lobo, Pilar" uniqKey="Blanco Lobo P" first="Pilar" last="Blanco-Lobo">Pilar Blanco-Lobo</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Reilly, Emma C" sort="Reilly, Emma C" uniqKey="Reilly E" first="Emma C." last="Reilly">Emma C. Reilly</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="af2-viruses-11-00928">David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Maehigashi, Tatsuya" sort="Maehigashi, Tatsuya" uniqKey="Maehigashi T" first="Tatsuya" last="Maehigashi">Tatsuya Maehigashi</name><affiliation><nlm:aff id="af3-viruses-11-00928">Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia, GA 30322, USA;<email>t.maehigashi@emory.edu</email> (T.M.);<email>baek.kim@emory.edu</email> (B.K.)</nlm:aff></affiliation></author><author><name sortKey="Nogales, Aitor" sort="Nogales, Aitor" uniqKey="Nogales A" first="Aitor" last="Nogales">Aitor Nogales</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Smith, Andrew" sort="Smith, Andrew" uniqKey="Smith A" first="Andrew" last="Smith">Andrew Smith</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="af4-viruses-11-00928">Medical Scientist Training Program (MSTP), University of Rochester, Rochester, New York, NY 14642, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Medical Scientist Training Program (MSTP), University of Rochester, Rochester, New York, NY 14642</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Topham, David J" sort="Topham, David J" uniqKey="Topham D" first="David J." last="Topham">David J. Topham</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="af2-viruses-11-00928">David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Dewhurst, Stephen" sort="Dewhurst, Stephen" uniqKey="Dewhurst S" first="Stephen" last="Dewhurst">Stephen Dewhurst</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author><author><name sortKey="Kim, Baek" sort="Kim, Baek" uniqKey="Kim B" first="Baek" last="Kim">Baek Kim</name><affiliation><nlm:aff id="af3-viruses-11-00928">Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia, GA 30322, USA;<email>t.maehigashi@emory.edu</email> (T.M.);<email>baek.kim@emory.edu</email> (B.K.)</nlm:aff></affiliation><affiliation wicri:level="1"><nlm:aff id="af5-viruses-11-00928">College of Pharmacy, Kyung-Hee University, Seoul 02447, Korea</nlm:aff><country xml:lang="fr">Corée du Sud</country><wicri:regionArea>College of Pharmacy, Kyung-Hee University, Seoul 02447</wicri:regionArea><placeName><settlement type="city">Séoul</settlement><region type="capital">Région capitale de Séoul</region></placeName></affiliation></author><author><name sortKey="Martinez Sobrido, Luis" sort="Martinez Sobrido, Luis" uniqKey="Martinez Sobrido L" first="Luis" last="Martínez-Sobrido">Luis Martínez-Sobrido</name><affiliation><nlm:aff id="af1-viruses-11-00928">Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</nlm:aff></affiliation></author></analytic><series><title level="j">Viruses</title><idno type="eISSN">1999-4915</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>Influenza viruses cause annual, seasonal infection across the globe. Vaccination represents the most effective strategy to prevent such infections and/or to reduce viral disease. Two major types of influenza vaccines are approved for human use: inactivated influenza vaccines (IIVs) and live attenuated influenza vaccines (LAIVs). Two Master Donor Virus (MDV) backbones have been used to create LAIVs against influenza A virus (IAV): the United States (US) A/Ann Arbor/6/60 (AA) and the Russian A/Leningrad/134/17/57 (Len) H2N2 viruses. The mutations responsible for the temperature sensitive (<italic>ts</italic>), cold-adapted (<italic>ca</italic>) and attenuated (<italic>att</italic>) phenotypes of the two MDVs have been previously identified and genetically mapped. However, a direct comparison of the contribution of these residues to viral attenuation, immunogenicity and protection efficacy has not been conducted. Here, we compared the In vitro and in vivo phenotype of recombinant influenza A/Puerto Rico/8/34 H1N1 (PR8) viruses containing the <italic>ts, ca</italic> and <italic>att</italic> mutations of the US (PR8/AA) and the Russian (PR8/Len) MDVs. Our results show that PR8/Len is more attenuated in vivo than PR8/AA, although both viruses induced similar levels of humoral and cellular responses, and protection against homologous and heterologous viral challenges. Our findings support the feasibility of using a different virus backbone as MDV for the development of improved LAIVs for the prevention of IAV infections.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Lynch, J P" uniqKey="Lynch J">J.P. Lynch</name></author><author><name sortKey="Walsh, E E" uniqKey="Walsh E">E.E. Walsh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barr, I G" uniqKey="Barr I">I.G. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Viruses</journal-id><journal-id journal-id-type="iso-abbrev">Viruses</journal-id><journal-id journal-id-type="publisher-id">viruses</journal-id><journal-title-group><journal-title>Viruses</journal-title></journal-title-group><issn pub-type="epub">1999-4915</issn><publisher><publisher-name>MDPI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31658679</article-id><article-id pub-id-type="pmc">6832241</article-id><article-id pub-id-type="doi">10.3390/v11100928</article-id><article-id pub-id-type="publisher-id">viruses-11-00928</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Rodriguez</surname><given-names>Laura</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref><xref ref-type="author-notes" rid="fn1-viruses-11-00928">†</xref></contrib><contrib contrib-type="author"><name><surname>Blanco-Lobo</surname><given-names>Pilar</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref></contrib><contrib contrib-type="author"><name><surname>Reilly</surname><given-names>Emma C.</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref><xref ref-type="aff" rid="af2-viruses-11-00928">2</xref></contrib><contrib contrib-type="author"><name><surname>Maehigashi</surname><given-names>Tatsuya</given-names></name><xref ref-type="aff" rid="af3-viruses-11-00928">3</xref></contrib><contrib contrib-type="author"><name><surname>Nogales</surname><given-names>Aitor</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref></contrib><contrib contrib-type="author"><name><surname>Smith</surname><given-names>Andrew</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref><xref ref-type="aff" rid="af4-viruses-11-00928">4</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0002-9435-8673</contrib-id><name><surname>Topham</surname><given-names>David J.</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref><xref ref-type="aff" rid="af2-viruses-11-00928">2</xref></contrib><contrib contrib-type="author"><name><surname>Dewhurst</surname><given-names>Stephen</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Baek</given-names></name><xref ref-type="aff" rid="af3-viruses-11-00928">3</xref><xref ref-type="aff" rid="af5-viruses-11-00928">5</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-7084-0804</contrib-id><name><surname>Martínez-Sobrido</surname><given-names>Luis</given-names></name><xref ref-type="aff" rid="af1-viruses-11-00928">1</xref><xref rid="c1-viruses-11-00928" ref-type="corresp">*</xref></contrib></contrib-group><aff id="af1-viruses-11-00928"><label>1</label>Department of Microbiology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA;<email>laurita85oviedo@hotmail.com</email> (L.R.);<email>piblanlo@gmail.com</email> (P.B.-L.);<email>emma_reilly@urmc.rochester.edu</email> (E.C.R.);<email>nogales.aitor@inia.es</email> (A.N.);<email>Andrew_Smith72@URMC.Rochester.edu</email> (A.S.);<email>David_topham@urmc.rochester.edu</email> (D.J.T.);<email>stephen_dewhurst@me.com</email> (S.D.)</aff><aff id="af2-viruses-11-00928"><label>2</label>David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York, NY 14642, USA</aff><aff id="af3-viruses-11-00928"><label>3</label>Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia, GA 30322, USA;<email>t.maehigashi@emory.edu</email> (T.M.);<email>baek.kim@emory.edu</email> (B.K.)</aff><aff id="af4-viruses-11-00928"><label>4</label>Medical Scientist Training Program (MSTP), University of Rochester, Rochester, New York, NY 14642, USA</aff><aff id="af5-viruses-11-00928"><label>5</label>College of Pharmacy, Kyung-Hee University, Seoul 02447, Korea</aff><author-notes><corresp id="c1-viruses-11-00928"><label>*</label>Correspondence: <email>luis_martinez@urmc.rochester.edu</email></corresp><fn id="fn1-viruses-11-00928"><label>†</label><p>Current address: Agencia Española de Medicamentos y Productos Sanitarios, E-28022 Madrid, Spain.</p></fn></author-notes><pub-date pub-type="epub"><day>10</day><month>10</month><year>2019</year></pub-date><pub-date pub-type="collection"><month>10</month><year>2019</year></pub-date><volume>11</volume><issue>10</issue><elocation-id>928</elocation-id><history><date date-type="received"><day>10</day><month>9</month><year>2019</year></date><date date-type="accepted"><day>08</day><month>10</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 by the authors.</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>Influenza viruses cause annual, seasonal infection across the globe. Vaccination represents the most effective strategy to prevent such infections and/or to reduce viral disease. Two major types of influenza vaccines are approved for human use: inactivated influenza vaccines (IIVs) and live attenuated influenza vaccines (LAIVs). Two Master Donor Virus (MDV) backbones have been used to create LAIVs against influenza A virus (IAV): the United States (US) A/Ann Arbor/6/60 (AA) and the Russian A/Leningrad/134/17/57 (Len) H2N2 viruses. The mutations responsible for the temperature sensitive (<italic>ts</italic>), cold-adapted (<italic>ca</italic>) and attenuated (<italic>att</italic>) phenotypes of the two MDVs have been previously identified and genetically mapped. However, a direct comparison of the contribution of these residues to viral attenuation, immunogenicity and protection efficacy has not been conducted. Here, we compared the In vitro and in vivo phenotype of recombinant influenza A/Puerto Rico/8/34 H1N1 (PR8) viruses containing the <italic>ts, ca</italic> and <italic>att</italic> mutations of the US (PR8/AA) and the Russian (PR8/Len) MDVs. Our results show that PR8/Len is more attenuated in vivo than PR8/AA, although both viruses induced similar levels of humoral and cellular responses, and protection against homologous and heterologous viral challenges. Our findings support the feasibility of using a different virus backbone as MDV for the development of improved LAIVs for the prevention of IAV infections.</p></abstract><kwd-group><kwd>influenza</kwd><kwd>vaccine</kwd><kwd>live-attenuated vaccine</kwd></kwd-group></article-meta></front><floats-group><fig id="viruses-11-00928-f001" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Effect of temperature on the polymerase activity of PR8/AA and PR8/Len. (<bold>A</bold>) Schematic representation of segments 1 (PB2), 2 (PB1) and 8 (NS) of PR8/WT (white, left), PR8/AA (black, middle) and PR8/Len (grey, right). Amino acid substitutions to generate PR8/AA and PR8/Len are indicated. B-C) Minigenome activity: HEK293T cells (12-well plate format, 4 × 10<sup>5</sup> cells/well, triplicates) were transiently co-transfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP) together with 500 ng of a vRNA-like expression plasmid encoding GFP (<bold>B</bold>) or Gluc (<bold>C</bold>) under the control of the human polymerase I promoter (hPol-I-GFP and -Gluc, respectively), and 100 ng of an SV40 Cluc expressing plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and viral replication and transcription was evaluated 24 h later by GFP (<bold>B</bold>) and luciferase (<bold>C</bold>) expression. Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that obtained in the absence of pDZ NP. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, <italic>P</italic> < 0.05; **, <italic>P</italic> < 0.01; NS, no statistical differences (Student <italic>t</italic> test). Scale bars, 200 μm.</p></caption><graphic xlink:href="viruses-11-00928-g001"></graphic></fig><fig id="viruses-11-00928-f002" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10<sup>5</sup> cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP (<bold>A</bold>) and -Gluc (<bold>B</bold>), and 100 ng of SV40 Cluc to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging (<bold>A</bold>) and Gluc expression (<bold>B</bold>). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, <italic>P</italic> < 0.05; **, <italic>P</italic> < 0.01; NS, no statistical differences (Student <italic>t</italic> test). Scale bars, 200 μm.</p></caption><graphic xlink:href="viruses-11-00928-g002"></graphic></fig><fig id="viruses-11-00928-f003" orientation="portrait" position="float"><label>Figure 3</label><caption><p>In vitro characterization of PR8/AA and PR8/Len viruses. (<bold>A</bold>) Multicycle growth kinetics: MDCK cells (12-well plate format, 4 × 10<sup>5</sup> cells/well, triplicates) were infected at low MOI (0.001) with PR8/WT (white bars), PR8/AA (black bars) and PR8/Len (grey bars) viruses and placed at 33 °C, 37 °C, or 39 °C. Tissue culture supernatants were recovered at the indicated times post-infection (p.i.) and viral titers were determined by immunofocus assay (FFU/mL) using an anti-NP mAb (HB-65). Data represent the means and SDs of the results determined from triplicate wells. Dotted black lines indicate the limit of detection (200 FFU/mL). (<bold>B</bold>) Plaque assays: MDCK cells (6-well plate format, 1 × 10<sup>6</sup> cells/well) were infected with PR8/WT, PR8/AA or PR8/Len viruses and incubated at 33 °C, 37 °C, or 39 °C for 3 days. Plaque phenotype was assessed by immunostaining with the HB-65 anti-NP mAb.</p></caption><graphic xlink:href="viruses-11-00928-g003"></graphic></fig><fig id="viruses-11-00928-f004" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Attenuation of PR8/AA and PR8/Len viruses in vivo. Six-to-8-week-old female C57BL/6 mice (<italic>n</italic> = 5) were infected intranasally (i.n.) with the indicated FFU of PR8/AA (<bold>A</bold>,<bold>B</bold>) or PR8/Len (<bold>C</bold>,<bold>D</bold>) viruses and then monitored daily for 2 weeks for body weight loss (<bold>A</bold>,<bold>C</bold>) and survival (<bold>B</bold>,<bold>D</bold>). Mice that lost 25% of their initial body weight were sacrificed. Data represent the means and SDs of the results determined for individual mice (<italic>n</italic> = 5). The viral 50% mouse lethal dose (MLD<sub>50</sub>) for PR8/AA and PR8/Len were calculated based on survival data using the method of Reed and Muench [<xref rid="B50-viruses-11-00928" ref-type="bibr">50</xref>]. (<bold>A</bold>,<bold>C</bold>) Statistical analysis was performed using one-way ANOVA test comparing % body weight loss between groups (* <italic>P</italic> < 0.0001). (<bold>B</bold>,<bold>D</bold>) Statistical analysis was performed using log-rank (Mantel-Cox) test (* <italic>P</italic> < 0.0001).</p></caption><graphic xlink:href="viruses-11-00928-g004"></graphic></fig><fig id="viruses-11-00928-f005" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Replication of PR8/AA and PR8/Len viruses in mice and induction of humoral immune responses. (<bold>A</bold>) Lung viral titers: Six-to-eight-week-old female C57BL/6 mice (<italic>n</italic> = 6) were infected i.n. with 10<sup>3</sup> and 10<sup>4</sup> FFU of PR8/Len and 10<sup>3</sup> FFU of PR8/AA viruses. Viral titer in lungs of infected mice was evaluated at days 2 and 4 p.i. (<italic>n</italic> = 3) by immunofocus assay (FFU/mL) in MDCK at 33 °C using an anti-NP mAb (HB-65). Data represent the means and SDs of the results determined from each group of mice (<italic>n</italic> = 3). Dotted black line represents the limit of detection of the assay (200 FFU/mL). NS, no statistical differences (Student <italic>t</italic> test). (<bold>B</bold>–<bold>D</bold>) Humoral immune responses: Six-to-eight-week-old female C57BL/6 mice (<italic>n</italic> = 5) were infected i.n. with 10<sup>3</sup> and 10<sup>4</sup> FFU of PR8/Len and 10<sup>3</sup> FFU of PR8/AA viruses, or mock-infected (PBS). At 14 days p.i., mice were bled and sera were collected and evaluated individually by HAI assay for hemagglutination inhibiting antibodies (<bold>B</bold>) and by ELISA for IgG Abs against total influenza viral proteins using cell extracts of MDCK cells infected with PR8/WT (<bold>C</bold>) or against recombinant purified PR8 HA protein (<bold>D</bold>). (<bold>B</bold>) HAI titers: ND, not detected. &, <italic>p</italic> < 0.05 (PR8/Len 10<sup>3</sup> FFU vs PR8/AA 10<sup>4</sup> FFU) (Student <italic>t</italic> test); NS, no statistical differences (Student <italic>t</italic> test). (<bold>C</bold>,<bold>D</bold>) ELISAs: OD, optical density. Data represent the means and SDs of the results for five individual mice. *, <italic>p</italic> < 0.001 (PR8/Len 10<sup>3</sup> FFU vs.PR8/AA 10<sup>3</sup> FFU); **, <italic>p</italic> < 0.05 (PR8/Len 10<sup>4</sup> FFU vs.PR8/AA 10<sup>3</sup> FFU); ***, <italic>p</italic> < 0.01 (PR8/Len 10<sup>3</sup> FFU vs.PR8/AA 10<sup>3</sup> FFU) (Student <italic>t</italic> test).</p></caption><graphic xlink:href="viruses-11-00928-g005"></graphic></fig><fig id="viruses-11-00928-f006" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Induction of CD8 T cell responses by PR8/AA and PR8/Len viruses: Female six-to-eight-week-old C57BL/6 mice (<italic>n</italic> = 5) were infected (i.n.) with 10<sup>3</sup> and 10<sup>4</sup> FFU of PR8/Len and 10<sup>3</sup> FFU of PR8/AA viruses. Mice were also mock (PBS) infected as an internal control. Ten days p.i., lungs (<bold>A</bold>–<bold>C</bold>) and spleens (<bold>D</bold>–<bold>F</bold>) were collected and resident cells were prepared for flow cytometry. Live CD8 T cells specific for NP (<bold>B</bold>,<bold>E</bold>) or PA (<bold>C</bold>,<bold>F</bold>) tetramers were counted. Data represent the means +/− SDs of the results for 5 individual mice. *, <italic>p</italic> < 0.001 for the indicated pair wise comparisons (Student <italic>t</italic> test).</p></caption><graphic xlink:href="viruses-11-00928-g006"></graphic></fig><fig id="viruses-11-00928-f007" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Protection efficacy of PR8/AA and PR8/Len against homologous viral challenge: Six-to-eight-week-old female C57BL/6 mice (<italic>n</italic> = 5) were infected i.n. with 10<sup>3</sup> and 10<sup>4</sup> FFU of PR8/Len and 10<sup>3</sup> FFU of PR8/AA viruses, or mock vaccinated (PBS). At 15 days p.i., mice were challenged i.n. with 1,000 MLD<sub>50</sub> of PR8/WT and monitored daily over 10 days for body weight loss (<bold>A</bold>) and survival (<bold>B</bold>). Mice that lost 25% of their initial body weight were sacrificed. Data represent the means and SDs of the results determined for individual mice (<italic>n</italic> = 5). Statistical analysis was performed using one-way ANOVA (<bold>A</bold>) and Dunnett’s (<bold>B</bold>) tests comparing the PBS group to the other groups (* <italic>P</italic> < 0.0001).</p></caption><graphic xlink:href="viruses-11-00928-g007"></graphic></fig><fig id="viruses-11-00928-f008" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Protection efficacy induced by PR8/AA and PR8/Len against heterologous virus challenge. Six-to-eight-week-old female C57BL/6 mice (<italic>n</italic> = 5) were infected i.n. with 10<sup>3</sup> and 10<sup>4</sup> FFU of PR8/Len or 10<sup>3</sup> FFU of PR8/AA viruses, or mock-infected (PBS). At 15 days p.i., mice were challenged i.n. with 50 MLD<sub>50</sub> of X-31 and then monitored daily over 10 days for body weight loss (<bold>A</bold>) and survival (<bold>B</bold>). Mice that lost 25% of their initial body weight were sacrificed. Data represent the means and SDs of the results determined for individual mice (<italic>n</italic> = 5). Statistical analysis was performed using one-way ANOVA (<bold>A</bold>) and Dunnett’s (<bold>B</bold>) tests comparing the PBS group to the other groups (* <italic>P</italic> < 0.0001).</p></caption><graphic xlink:href="viruses-11-00928-g008"></graphic></fig><fig id="viruses-11-00928-f009" orientation="portrait" position="float"><label>Figure 9</label><caption><p>Location of the <italic>ts, ca</italic> and <italic>att</italic> mutations of AA and Len MDV in the viral polymerase complex. (<bold>A</bold>) PB1 (green spheres) and PB2 (purple sphere) mutations responsible for the ts, ca and att phenotype of AA are shown on the reference model of the RNA dependent RNA polymerase, RdRp, complex (PDB ID 5M3H) [<xref rid="B58-viruses-11-00928" ref-type="bibr">58</xref>]. The RdRp complex is shown as a surface-rendering model with its subunits PA (blue), PB1 (yellow) and PB2 (purple), and bound vRNA (red). Dotted square (red) indicates the PB2 cap-binding domain. Red asterisk shows the approximate location of the endonuclease catalytic site of PA. (<bold>B</bold>) Close-up images of the locations of the AA PB1 mutations E581G and A661T (left) and K391E (right). (<bold>C</bold>) Same as in (<bold>A</bold>) but for Len. (<bold>D</bold>) Close up locations of LenV591I (PB1, left), K265N (PB1, right) and V478L (PB2, right) mutations. All images were generated and rendered in the program Pymol [<xref rid="B59-viruses-11-00928" ref-type="bibr">59</xref>].</p></caption><graphic xlink:href="viruses-11-00928-g009"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Corée du Sud</li><li>États-Unis</li></country><region><li>Région capitale de Séoul</li><li>État de New York</li></region><settlement><li>Séoul</li></settlement></list><tree><noCountry><name sortKey="Blanco Lobo, Pilar" sort="Blanco Lobo, Pilar" uniqKey="Blanco Lobo P" first="Pilar" last="Blanco-Lobo">Pilar Blanco-Lobo</name><name sortKey="Dewhurst, Stephen" sort="Dewhurst, Stephen" uniqKey="Dewhurst S" first="Stephen" last="Dewhurst">Stephen Dewhurst</name><name sortKey="Maehigashi, Tatsuya" sort="Maehigashi, Tatsuya" uniqKey="Maehigashi T" first="Tatsuya" last="Maehigashi">Tatsuya Maehigashi</name><name sortKey="Martinez Sobrido, Luis" sort="Martinez Sobrido, Luis" uniqKey="Martinez Sobrido L" first="Luis" last="Martínez-Sobrido">Luis Martínez-Sobrido</name><name sortKey="Nogales, Aitor" sort="Nogales, Aitor" uniqKey="Nogales A" first="Aitor" last="Nogales">Aitor Nogales</name><name sortKey="Rodriguez, Laura" sort="Rodriguez, Laura" uniqKey="Rodriguez L" first="Laura" last="Rodriguez">Laura Rodriguez</name></noCountry><country name="États-Unis"><region name="État de New York"><name sortKey="Reilly, Emma C" sort="Reilly, Emma C" uniqKey="Reilly E" first="Emma C." last="Reilly">Emma C. Reilly</name></region><name sortKey="Smith, Andrew" sort="Smith, Andrew" uniqKey="Smith A" first="Andrew" last="Smith">Andrew Smith</name><name sortKey="Topham, David J" sort="Topham, David J" uniqKey="Topham D" first="David J." last="Topham">David J. Topham</name></country><country name="Corée du Sud"><region name="Région capitale de Séoul"><name sortKey="Kim, Baek" sort="Kim, Baek" uniqKey="Kim B" first="Baek" last="Kim">Baek Kim</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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