Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments—The importance of immunofocusing in subunit vaccine design</title>
<author><name sortKey="Ma, Cuiqing" sort="Ma, Cuiqing" uniqKey="Ma C" first="Cuiqing" last="Ma">Cuiqing Ma</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Lili" sort="Wang, Lili" uniqKey="Wang L" first="Lili" last="Wang">Lili Wang</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tao, Xinrong" sort="Tao, Xinrong" uniqKey="Tao X" first="Xinrong" last="Tao">Xinrong Tao</name>
<affiliation><nlm:aff id="aff0010">Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhang, Naru" sort="Zhang, Naru" uniqKey="Zhang N" first="Naru" last="Zhang">Naru Zhang</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yang, Yang" sort="Yang, Yang" uniqKey="Yang Y" first="Yang" last="Yang">Yang Yang</name>
<affiliation><nlm:aff id="aff0015">Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tseng, Chien Te K" sort="Tseng, Chien Te K" uniqKey="Tseng C" first="Chien-Te K" last="Tseng">Chien-Te K. Tseng</name>
<affiliation><nlm:aff id="aff0010">Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name>
<affiliation><nlm:aff id="aff0015">Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhou, Yusen" sort="Zhou, Yusen" uniqKey="Zhou Y" first="Yusen" last="Zhou">Yusen Zhou</name>
<affiliation><nlm:aff id="aff0020">State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff0025">Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">25240756</idno>
<idno type="pmc">4194190</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194190</idno>
<idno type="RBID">PMC:4194190</idno>
<idno type="doi">10.1016/j.vaccine.2014.08.086</idno>
<date when="2014">2014</date>
<idno type="wicri:Area/Pmc/Corpus">001165</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001165</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments—The importance of immunofocusing in subunit vaccine design</title>
<author><name sortKey="Ma, Cuiqing" sort="Ma, Cuiqing" uniqKey="Ma C" first="Cuiqing" last="Ma">Cuiqing Ma</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Lili" sort="Wang, Lili" uniqKey="Wang L" first="Lili" last="Wang">Lili Wang</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tao, Xinrong" sort="Tao, Xinrong" uniqKey="Tao X" first="Xinrong" last="Tao">Xinrong Tao</name>
<affiliation><nlm:aff id="aff0010">Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhang, Naru" sort="Zhang, Naru" uniqKey="Zhang N" first="Naru" last="Zhang">Naru Zhang</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yang, Yang" sort="Yang, Yang" uniqKey="Yang Y" first="Yang" last="Yang">Yang Yang</name>
<affiliation><nlm:aff id="aff0015">Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tseng, Chien Te K" sort="Tseng, Chien Te K" uniqKey="Tseng C" first="Chien-Te K" last="Tseng">Chien-Te K. Tseng</name>
<affiliation><nlm:aff id="aff0010">Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name>
<affiliation><nlm:aff id="aff0015">Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhou, Yusen" sort="Zhou, Yusen" uniqKey="Zhou Y" first="Yusen" last="Zhou">Yusen Zhou</name>
<affiliation><nlm:aff id="aff0020">State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff0025">Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name>
<affiliation><nlm:aff id="aff0005">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Vaccine</title>
<idno type="ISSN">0264-410X</idno>
<idno type="eISSN">1873-2518</idno>
<imprint><date when="2014">2014</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><title>Highlights</title>
<p><list list-type="simple" id="lis0005"><list-item id="lsti0005"><label>•</label>
<p id="par0005">We express five MERS-CoV RBDs spanning S350-588, 358-588, 367-588, 377-606, and 377-588.</p>
</list-item>
<list-item id="lsti0010"><label>•</label>
<p id="par0010">S377-588, 367-588 and 358-588 have the highest receptor binding ability than others.</p>
</list-item>
<list-item id="lsti0015"><label>•</label>
<p id="par0015">S377-588 and 367-588 induced the highest-titer of IgG antibodies in mice.</p>
</list-item>
<list-item id="lsti0020"><label>•</label>
<p id="par0020">S377-588 elicits highest neutralization in mice and high neutralization in rabbits.</p>
</list-item>
<list-item id="lsti0025"><label>•</label>
<p id="par0025">S377-588 contains stably folded RBD structure and major neutralizing epitopes.</p>
</list-item>
</list>
</p>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zaki, A M" uniqKey="Zaki A">A.M. Zaki</name>
</author>
<author><name sortKey="Van, B S" uniqKey="Van B">B.S. van</name>
</author>
<author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T.M. Bestebroer</name>
</author>
<author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A.D. Osterhaus</name>
</author>
<author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R.A. Fouchier</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Gulland, A" uniqKey="Gulland A">A. Gulland</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kupferschmidt, K" uniqKey="Kupferschmidt K">K. Kupferschmidt</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Memish, Z A" uniqKey="Memish Z">Z.A. Memish</name>
</author>
<author><name sortKey="Mishra, N" uniqKey="Mishra N">N. Mishra</name>
</author>
<author><name sortKey="Olival, K J" uniqKey="Olival K">K.J. Olival</name>
</author>
<author><name sortKey="Fagbo, S F" uniqKey="Fagbo S">S.F. Fagbo</name>
</author>
<author><name sortKey="Kapoor, V" uniqKey="Kapoor V">V. Kapoor</name>
</author>
<author><name sortKey="Epstein, J H" uniqKey="Epstein J">J.H. Epstein</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Perera, R A" uniqKey="Perera R">R.A. Perera</name>
</author>
<author><name sortKey="Wang, P" uniqKey="Wang P">P. Wang</name>
</author>
<author><name sortKey="Gomaa, M R" uniqKey="Gomaa M">M.R. Gomaa</name>
</author>
<author><name sortKey="El Shesheny, R" uniqKey="El Shesheny R">R. El-Shesheny</name>
</author>
<author><name sortKey="Kandeil, A" uniqKey="Kandeil A">A. Kandeil</name>
</author>
<author><name sortKey="Bagato, O" uniqKey="Bagato O">O. Bagato</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Reusken, C B" uniqKey="Reusken C">C.B. Reusken</name>
</author>
<author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B.L. Haagmans</name>
</author>
<author><name sortKey="Muller, M A" uniqKey="Muller M">M.A. Muller</name>
</author>
<author><name sortKey="Gutierrez, C" uniqKey="Gutierrez C">C. Gutierrez</name>
</author>
<author><name sortKey="Godeke, G J" uniqKey="Godeke G">G.J. Godeke</name>
</author>
<author><name sortKey="Meyer, B" uniqKey="Meyer B">B. Meyer</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Azhar, E I" uniqKey="Azhar E">E.I. Azhar</name>
</author>
<author><name sortKey="El Kafrawy, S A" uniqKey="El Kafrawy S">S.A. El-Kafrawy</name>
</author>
<author><name sortKey="Farraj, S A" uniqKey="Farraj S">S.A. Farraj</name>
</author>
<author><name sortKey="Hassan, A M" uniqKey="Hassan A">A.M. Hassan</name>
</author>
<author><name sortKey="Al Saeed, M S" uniqKey="Al Saeed M">M.S. Al-Saeed</name>
</author>
<author><name sortKey="Hashem, A M" uniqKey="Hashem A">A.M. Hashem</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Chu, D K" uniqKey="Chu D">D.K. Chu</name>
</author>
<author><name sortKey="Poon, L L" uniqKey="Poon L">L.L. Poon</name>
</author>
<author><name sortKey="Gomaa, M M" uniqKey="Gomaa M">M.M. Gomaa</name>
</author>
<author><name sortKey="Shehata, M M" uniqKey="Shehata M">M.M. Shehata</name>
</author>
<author><name sortKey="Perera, R A" uniqKey="Perera R">R.A. Perera</name>
</author>
<author><name sortKey="Abu, Z D" uniqKey="Abu Z">Z.D. Abu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Meyer, B" uniqKey="Meyer B">B. Meyer</name>
</author>
<author><name sortKey="Muller, M A" uniqKey="Muller M">M.A. Muller</name>
</author>
<author><name sortKey="Corman, V M" uniqKey="Corman V">V.M. Corman</name>
</author>
<author><name sortKey="Reusken, C B" uniqKey="Reusken C">C.B. Reusken</name>
</author>
<author><name sortKey="Ritz, D" uniqKey="Ritz D">D. Ritz</name>
</author>
<author><name sortKey="Godeke, G J" uniqKey="Godeke G">G.J. Godeke</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Briese, T" uniqKey="Briese T">T. Briese</name>
</author>
<author><name sortKey="Mishra, N" uniqKey="Mishra N">N. Mishra</name>
</author>
<author><name sortKey="Jain, K" uniqKey="Jain K">K. Jain</name>
</author>
<author><name sortKey="Zalmout, I S" uniqKey="Zalmout I">I.S. Zalmout</name>
</author>
<author><name sortKey="Jabado, O J" uniqKey="Jabado O">O.J. Jabado</name>
</author>
<author><name sortKey="Karesh, W B" uniqKey="Karesh W">W.B. Karesh</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Alagaili, A N" uniqKey="Alagaili A">A.N. Alagaili</name>
</author>
<author><name sortKey="Briese, T" uniqKey="Briese T">T. Briese</name>
</author>
<author><name sortKey="Mishra, N" uniqKey="Mishra N">N. Mishra</name>
</author>
<author><name sortKey="Kapoor, V" uniqKey="Kapoor V">V. Kapoor</name>
</author>
<author><name sortKey="Sameroff, S C" uniqKey="Sameroff S">S.C. Sameroff</name>
</author>
<author><name sortKey="Burbelo, P D" uniqKey="Burbelo P">P.D. Burbelo</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Mailles, A" uniqKey="Mailles A">A. Mailles</name>
</author>
<author><name sortKey="Blanckaert, K" uniqKey="Blanckaert K">K. Blanckaert</name>
</author>
<author><name sortKey="Chaud, P" uniqKey="Chaud P">P. Chaud</name>
</author>
<author><name sortKey="Van Der Werf, S" uniqKey="Van Der Werf S">S. van der Werf</name>
</author>
<author><name sortKey="Lina, B" uniqKey="Lina B">B. Lina</name>
</author>
<author><name sortKey="Caro, V" uniqKey="Caro V">V. Caro</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Puzelli, S" uniqKey="Puzelli S">S. Puzelli</name>
</author>
<author><name sortKey="Azzi, A" uniqKey="Azzi A">A. Azzi</name>
</author>
<author><name sortKey="Santini, M G" uniqKey="Santini M">M.G. Santini</name>
</author>
<author><name sortKey="Di, M A" uniqKey="Di M">M.A. Di</name>
</author>
<author><name sortKey="Facchini, M" uniqKey="Facchini M">M. Facchini</name>
</author>
<author><name sortKey="Castrucci, M R" uniqKey="Castrucci M">M.R. Castrucci</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Li, F" uniqKey="Li F">F. Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Raj, V S" uniqKey="Raj V">V.S. Raj</name>
</author>
<author><name sortKey="Mou, H" uniqKey="Mou H">H. Mou</name>
</author>
<author><name sortKey="Smits, S L" uniqKey="Smits S">S.L. Smits</name>
</author>
<author><name sortKey="Dekkers, D H" uniqKey="Dekkers D">D.H. Dekkers</name>
</author>
<author><name sortKey="Muller, M A" uniqKey="Muller M">M.A. Muller</name>
</author>
<author><name sortKey="Dijkman, R" uniqKey="Dijkman R">R. Dijkman</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lu, G" uniqKey="Lu G">G. Lu</name>
</author>
<author><name sortKey="Hu, Y" uniqKey="Hu Y">Y. Hu</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Qi, J" uniqKey="Qi J">J. Qi</name>
</author>
<author><name sortKey="Gao, F" uniqKey="Gao F">F. Gao</name>
</author>
<author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Mou, H" uniqKey="Mou H">H. Mou</name>
</author>
<author><name sortKey="Raj, V S" uniqKey="Raj V">V.S. Raj</name>
</author>
<author><name sortKey="Van Kuppeveld, F J" uniqKey="Van Kuppeveld F">F.J. van Kuppeveld</name>
</author>
<author><name sortKey="Rottier, P J" uniqKey="Rottier P">P.J. Rottier</name>
</author>
<author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B.L. Haagmans</name>
</author>
<author><name sortKey="Bosch, B J" uniqKey="Bosch B">B.J. Bosch</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Kou, Z" uniqKey="Kou Z">Z. Kou</name>
</author>
<author><name sortKey="Ma, C" uniqKey="Ma C">C. Ma</name>
</author>
<author><name sortKey="Tao, X" uniqKey="Tao X">X. Tao</name>
</author>
<author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Chen, Y" uniqKey="Chen Y">Y. Chen</name>
</author>
<author><name sortKey="Rajashankar, K R" uniqKey="Rajashankar K">K.R. Rajashankar</name>
</author>
<author><name sortKey="Yang, Y" uniqKey="Yang Y">Y. Yang</name>
</author>
<author><name sortKey="Agnihothram, S S" uniqKey="Agnihothram S">S.S. Agnihothram</name>
</author>
<author><name sortKey="Liu, C" uniqKey="Liu C">C. Liu</name>
</author>
<author><name sortKey="Lin, Y L" uniqKey="Lin Y">Y.L. Lin</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wang, N" uniqKey="Wang N">N. Wang</name>
</author>
<author><name sortKey="Shi, X" uniqKey="Shi X">X. Shi</name>
</author>
<author><name sortKey="Jiang, L" uniqKey="Jiang L">L. Jiang</name>
</author>
<author><name sortKey="Zhang, S" uniqKey="Zhang S">S. Zhang</name>
</author>
<author><name sortKey="Wang, D" uniqKey="Wang D">D. Wang</name>
</author>
<author><name sortKey="Tong, P" uniqKey="Tong P">P. Tong</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhong, N S" uniqKey="Zhong N">N.S. Zhong</name>
</author>
<author><name sortKey="Zheng, B J" uniqKey="Zheng B">B.J. Zheng</name>
</author>
<author><name sortKey="Li, Y M" uniqKey="Li Y">Y.M. Li</name>
</author>
<author><name sortKey="Poon" uniqKey="Poon">Poon</name>
</author>
<author><name sortKey="Xie, Z H" uniqKey="Xie Z">Z.H. Xie</name>
</author>
<author><name sortKey="Chan, K H" uniqKey="Chan K">K.H. Chan</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Skowronski, D M" uniqKey="Skowronski D">D.M. Skowronski</name>
</author>
<author><name sortKey="Astell, C" uniqKey="Astell C">C. Astell</name>
</author>
<author><name sortKey="Brunham, R C" uniqKey="Brunham R">R.C. Brunham</name>
</author>
<author><name sortKey="Low, D E" uniqKey="Low D">D.E. Low</name>
</author>
<author><name sortKey="Petric, M" uniqKey="Petric M">M. Petric</name>
</author>
<author><name sortKey="Roper, R L" uniqKey="Roper R">R.L. Roper</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="He, Y" uniqKey="He Y">Y. He</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
<author><name sortKey="Liu, S" uniqKey="Liu S">S. Liu</name>
</author>
<author><name sortKey="Zheng, B J" uniqKey="Zheng B">B.J. Zheng</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Chan, C C" uniqKey="Chan C">C.C. Chan</name>
</author>
<author><name sortKey="Sun, S" uniqKey="Sun S">S. Sun</name>
</author>
<author><name sortKey="Chen, M" uniqKey="Chen M">M. Chen</name>
</author>
<author><name sortKey="Liu, Z" uniqKey="Liu Z">Z. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="He, Y" uniqKey="He Y">Y. He</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
<author><name sortKey="Liu, S" uniqKey="Liu S">S. Liu</name>
</author>
<author><name sortKey="Kou, Z" uniqKey="Kou Z">Z. Kou</name>
</author>
<author><name sortKey="Li, W" uniqKey="Li W">W. Li</name>
</author>
<author><name sortKey="Farzan, M" uniqKey="Farzan M">M. Farzan</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="He, Y" uniqKey="He Y">Y. He</name>
</author>
<author><name sortKey="Lu, H" uniqKey="Lu H">H. Lu</name>
</author>
<author><name sortKey="Siddiqui, P" uniqKey="Siddiqui P">P. Siddiqui</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Chan, C C" uniqKey="Chan C">C.C. Chan</name>
</author>
<author><name sortKey="Li, L" uniqKey="Li L">L. Li</name>
</author>
<author><name sortKey="He, Y" uniqKey="He Y">Y. He</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Li, F" uniqKey="Li F">F. Li</name>
</author>
<author><name sortKey="Li, W" uniqKey="Li W">W. Li</name>
</author>
<author><name sortKey="Farzan, M" uniqKey="Farzan M">M. Farzan</name>
</author>
<author><name sortKey="Harrison, S C" uniqKey="Harrison S">S.C. Harrison</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Kou, Z" uniqKey="Kou Z">Z. Kou</name>
</author>
<author><name sortKey="Ma, C" uniqKey="Ma C">C. Ma</name>
</author>
<author><name sortKey="Sun, S" uniqKey="Sun S">S. Sun</name>
</author>
<author><name sortKey="Poon, V K" uniqKey="Poon V">V.K. Poon</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Ma, C" uniqKey="Ma C">C. Ma</name>
</author>
<author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name>
</author>
<author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Tao, X" uniqKey="Tao X">X. Tao</name>
</author>
<author><name sortKey="Tseng, C T" uniqKey="Tseng C">C.T. Tseng</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Yang, Y" uniqKey="Yang Y">Y. Yang</name>
</author>
<author><name sortKey="Qiu, H" uniqKey="Qiu H">H. Qiu</name>
</author>
<author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name>
</author>
<author><name sortKey="Kou, Z" uniqKey="Kou Z">Z. Kou</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Tao, X" uniqKey="Tao X">X. Tao</name>
</author>
<author><name sortKey="Hill, T E" uniqKey="Hill T">T.E. Hill</name>
</author>
<author><name sortKey="Morimoto, C" uniqKey="Morimoto C">C. Morimoto</name>
</author>
<author><name sortKey="Peters, C J" uniqKey="Peters C">C.J. Peters</name>
</author>
<author><name sortKey="Ksiazek, T G" uniqKey="Ksiazek T">T.G. Ksiazek</name>
</author>
<author><name sortKey="Tseng, C T" uniqKey="Tseng C">C.T. Tseng</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhang, N" uniqKey="Zhang N">N. Zhang</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Song, F" uniqKey="Song F">F. Song</name>
</author>
<author><name sortKey="Fux, R" uniqKey="Fux R">R. Fux</name>
</author>
<author><name sortKey="Provacia, L B" uniqKey="Provacia L">L.B. Provacia</name>
</author>
<author><name sortKey="Volz, A" uniqKey="Volz A">A. Volz</name>
</author>
<author><name sortKey="Eickmann, M" uniqKey="Eickmann M">M. Eickmann</name>
</author>
<author><name sortKey="Becker, S" uniqKey="Becker S">S. Becker</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Agnihothram, S" uniqKey="Agnihothram S">S. Agnihothram</name>
</author>
<author><name sortKey="Gopal, R" uniqKey="Gopal R">R. Gopal</name>
</author>
<author><name sortKey="Yount, B L" uniqKey="Yount B">B.L. Yount</name>
</author>
<author><name sortKey="Donaldson, E F" uniqKey="Donaldson E">E.F. Donaldson</name>
</author>
<author><name sortKey="Menachery, V D" uniqKey="Menachery V">V.D. Menachery</name>
</author>
<author><name sortKey="Graham, R L" uniqKey="Graham R">R.L. Graham</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Coleman, C M" uniqKey="Coleman C">C.M. Coleman</name>
</author>
<author><name sortKey="Liu, Y V" uniqKey="Liu Y">Y.V. Liu</name>
</author>
<author><name sortKey="Mu, H" uniqKey="Mu H">H. Mu</name>
</author>
<author><name sortKey="Taylor, J K" uniqKey="Taylor J">J.K. Taylor</name>
</author>
<author><name sortKey="Massare, M" uniqKey="Massare M">M. Massare</name>
</author>
<author><name sortKey="Flyer, D C" uniqKey="Flyer D">D.C. Flyer</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Martyn, J C" uniqKey="Martyn J">J.C. Martyn</name>
</author>
<author><name sortKey="Cardin, A J" uniqKey="Cardin A">A.J. Cardin</name>
</author>
<author><name sortKey="Wines, B D" uniqKey="Wines B">B.D. Wines</name>
</author>
<author><name sortKey="Cendron, A" uniqKey="Cendron A">A. Cendron</name>
</author>
<author><name sortKey="Li, S" uniqKey="Li S">S. Li</name>
</author>
<author><name sortKey="Mackenzie, J" uniqKey="Mackenzie J">J. Mackenzie</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name>
</author>
<author><name sortKey="Xu, X" uniqKey="Xu X">X. Xu</name>
</author>
<author><name sortKey="Jones, I M" uniqKey="Jones I">I.M. Jones</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Sun, S" uniqKey="Sun S">S. Sun</name>
</author>
<author><name sortKey="Zhang, X" uniqKey="Zhang X">X. Zhang</name>
</author>
<author><name sortKey="Zhou, X" uniqKey="Zhou X">X. Zhou</name>
</author>
<author><name sortKey="Guo, Y" uniqKey="Guo Y">Y. Guo</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Leung, V H" uniqKey="Leung V">V.H. Leung</name>
</author>
<author><name sortKey="Zhang, X" uniqKey="Zhang X">X. Zhang</name>
</author>
<author><name sortKey="Zhou, J" uniqKey="Zhou J">J. Zhou</name>
</author>
<author><name sortKey="Chen, M" uniqKey="Chen M">M. Chen</name>
</author>
<author><name sortKey="He, W" uniqKey="He W">W. He</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="He, Y" uniqKey="He Y">Y. He</name>
</author>
<author><name sortKey="Guo, Y" uniqKey="Guo Y">Y. Guo</name>
</author>
<author><name sortKey="Zheng, B J" uniqKey="Zheng B">B.J. Zheng</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Qiu, H" uniqKey="Qiu H">H. Qiu</name>
</author>
<author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Jiang, L" uniqKey="Jiang L">L. Jiang</name>
</author>
<author><name sortKey="Wang, N" uniqKey="Wang N">N. Wang</name>
</author>
<author><name sortKey="Zuo, T" uniqKey="Zuo T">T. Zuo</name>
</author>
<author><name sortKey="Shi, X" uniqKey="Shi X">X. Shi</name>
</author>
<author><name sortKey="Poon, K M" uniqKey="Poon K">K.M. Poon</name>
</author>
<author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Tang, X C" uniqKey="Tang X">X.C. Tang</name>
</author>
<author><name sortKey="Agnihothram, S S" uniqKey="Agnihothram S">S.S. Agnihothram</name>
</author>
<author><name sortKey="Jiao, Y" uniqKey="Jiao Y">Y. Jiao</name>
</author>
<author><name sortKey="Stanhope, J" uniqKey="Stanhope J">J. Stanhope</name>
</author>
<author><name sortKey="Graham, R L" uniqKey="Graham R">R.L. Graham</name>
</author>
<author><name sortKey="Peterson, E C" uniqKey="Peterson E">E.C. Peterson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Ying, T" uniqKey="Ying T">T. Ying</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Ju, T W" uniqKey="Ju T">T.W. Ju</name>
</author>
<author><name sortKey="Prabakaran, P" uniqKey="Prabakaran P">P. Prabakaran</name>
</author>
<author><name sortKey="Lau, C C" uniqKey="Lau C">C.C. Lau</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Czub, M" uniqKey="Czub M">M. Czub</name>
</author>
<author><name sortKey="Weingartl, H" uniqKey="Weingartl H">H. Weingartl</name>
</author>
<author><name sortKey="Czub, S" uniqKey="Czub S">S. Czub</name>
</author>
<author><name sortKey="He, R" uniqKey="He R">R. He</name>
</author>
<author><name sortKey="Cao, J" uniqKey="Cao J">J. Cao</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="He, Y" uniqKey="He Y">Y. He</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
<author><name sortKey="Wu, H" uniqKey="Wu H">H. Wu</name>
</author>
<author><name sortKey="Luo, B" uniqKey="Luo B">B. Luo</name>
</author>
<author><name sortKey="Chen, J" uniqKey="Chen J">J. Chen</name>
</author>
<author><name sortKey="Li, W" uniqKey="Li W">W. Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Weingartl, H" uniqKey="Weingartl H">H. Weingartl</name>
</author>
<author><name sortKey="Czub, M" uniqKey="Czub M">M. Czub</name>
</author>
<author><name sortKey="Czub, S" uniqKey="Czub S">S. Czub</name>
</author>
<author><name sortKey="Neufeld, J" uniqKey="Neufeld J">J. Neufeld</name>
</author>
<author><name sortKey="Marszal, P" uniqKey="Marszal P">P. Marszal</name>
</author>
<author><name sortKey="Gren, J" uniqKey="Gren J">J. Gren</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="De, W E" uniqKey="De W">W.E. de</name>
</author>
<author><name sortKey="Rasmussen, A L" uniqKey="Rasmussen A">A.L. Rasmussen</name>
</author>
<author><name sortKey="Falzarano, D" uniqKey="Falzarano D">D. Falzarano</name>
</author>
<author><name sortKey="Bushmaker, T" uniqKey="Bushmaker T">T. Bushmaker</name>
</author>
<author><name sortKey="Feldmann, F" uniqKey="Feldmann F">F. Feldmann</name>
</author>
<author><name sortKey="Brining, D L" uniqKey="Brining D">D.L. Brining</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Yao, Y" uniqKey="Yao Y">Y. Yao</name>
</author>
<author><name sortKey="Bao, L" uniqKey="Bao L">L. Bao</name>
</author>
<author><name sortKey="Deng, W" uniqKey="Deng W">W. Deng</name>
</author>
<author><name sortKey="Xu, L" uniqKey="Xu L">L. Xu</name>
</author>
<author><name sortKey="Li, F" uniqKey="Li F">F. Li</name>
</author>
<author><name sortKey="Lv, Q" uniqKey="Lv Q">Q. Lv</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="De, W E" uniqKey="De W">W.E. de</name>
</author>
<author><name sortKey="Prescott, J" uniqKey="Prescott J">J. Prescott</name>
</author>
<author><name sortKey="Baseler, L" uniqKey="Baseler L">L. Baseler</name>
</author>
<author><name sortKey="Bushmaker, T" uniqKey="Bushmaker T">T. Bushmaker</name>
</author>
<author><name sortKey="Thomas, T" uniqKey="Thomas T">T. Thomas</name>
</author>
<author><name sortKey="Lackemeyer, M G" uniqKey="Lackemeyer M">M.G. Lackemeyer</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Coleman, C M" uniqKey="Coleman C">C.M. Coleman</name>
</author>
<author><name sortKey="Matthews, K L" uniqKey="Matthews K">K.L. Matthews</name>
</author>
<author><name sortKey="Goicochea, L" uniqKey="Goicochea L">L. Goicochea</name>
</author>
<author><name sortKey="Frieman, M B" uniqKey="Frieman M">M.B. Frieman</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Martina, B E" uniqKey="Martina B">B.E. Martina</name>
</author>
<author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B.L. Haagmans</name>
</author>
<author><name sortKey="Kuiken, T" uniqKey="Kuiken T">T. Kuiken</name>
</author>
<author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R.A. Fouchier</name>
</author>
<author><name sortKey="Rimmelzwaan, G F" uniqKey="Rimmelzwaan G">G.F. Rimmelzwaan</name>
</author>
<author><name sortKey="Van, A G" uniqKey="Van A">A.G. Van</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Roberts, A" uniqKey="Roberts A">A. Roberts</name>
</author>
<author><name sortKey="Vogel, L" uniqKey="Vogel L">L. Vogel</name>
</author>
<author><name sortKey="Guarner, J" uniqKey="Guarner J">J. Guarner</name>
</author>
<author><name sortKey="Hayes, N" uniqKey="Hayes N">N. Hayes</name>
</author>
<author><name sortKey="Murphy, B" uniqKey="Murphy B">B. Murphy</name>
</author>
<author><name sortKey="Zaki, S" uniqKey="Zaki S">S. Zaki</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhao, J" uniqKey="Zhao J">J. Zhao</name>
</author>
<author><name sortKey="Li, K" uniqKey="Li K">K. Li</name>
</author>
<author><name sortKey="Wohlford Lenane, C" uniqKey="Wohlford Lenane C">C. Wohlford-Lenane</name>
</author>
<author><name sortKey="Agnihothram, S S" uniqKey="Agnihothram S">S.S. Agnihothram</name>
</author>
<author><name sortKey="Fett, C" uniqKey="Fett C">C. Fett</name>
</author>
<author><name sortKey="Zhao, J" uniqKey="Zhao J">J. Zhao</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Vaccine</journal-id>
<journal-id journal-id-type="iso-abbrev">Vaccine</journal-id>
<journal-title-group><journal-title>Vaccine</journal-title>
</journal-title-group>
<issn pub-type="ppub">0264-410X</issn>
<issn pub-type="epub">1873-2518</issn>
<publisher><publisher-name>Elsevier Ltd.</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">25240756</article-id>
<article-id pub-id-type="pmc">4194190</article-id>
<article-id pub-id-type="publisher-id">S0264-410X(14)01255-9</article-id>
<article-id pub-id-type="doi">10.1016/j.vaccine.2014.08.086</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments—The importance of immunofocusing in subunit vaccine design</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="aut0005"><name><surname>Ma</surname>
<given-names>Cuiqing</given-names>
</name>
<xref rid="aff0005" ref-type="aff">a</xref>
<xref rid="fn1" ref-type="fn">1</xref>
</contrib>
<contrib contrib-type="author" id="aut0010"><name><surname>Wang</surname>
<given-names>Lili</given-names>
</name>
<xref rid="aff0005" ref-type="aff">a</xref>
<xref rid="fn1" ref-type="fn">1</xref>
</contrib>
<contrib contrib-type="author" id="aut0015"><name><surname>Tao</surname>
<given-names>Xinrong</given-names>
</name>
<xref rid="aff0010" ref-type="aff">b</xref>
</contrib>
<contrib contrib-type="author" id="aut0020"><name><surname>Zhang</surname>
<given-names>Naru</given-names>
</name>
<xref rid="aff0005" ref-type="aff">a</xref>
</contrib>
<contrib contrib-type="author" id="aut0025"><name><surname>Yang</surname>
<given-names>Yang</given-names>
</name>
<xref rid="aff0015" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="aut0030"><name><surname>Tseng</surname>
<given-names>Chien-Te K</given-names>
</name>
<xref rid="aff0010" ref-type="aff">b</xref>
</contrib>
<contrib contrib-type="author" id="aut0035"><name><surname>Li</surname>
<given-names>Fang</given-names>
</name>
<xref rid="aff0015" ref-type="aff">c</xref>
</contrib>
<contrib contrib-type="author" id="aut0040"><name><surname>Zhou</surname>
<given-names>Yusen</given-names>
</name>
<xref rid="aff0020" ref-type="aff">d</xref>
</contrib>
<contrib contrib-type="author" id="aut0045"><name><surname>Jiang</surname>
<given-names>Shibo</given-names>
</name>
<email>sjiang@nybloodcenter.org</email>
<xref rid="aff0005" ref-type="aff">a</xref>
<xref rid="aff0025" ref-type="aff">e</xref>
<xref rid="cor0005" ref-type="corresp">⁎</xref>
</contrib>
<contrib contrib-type="author" id="aut0050"><name><surname>Du</surname>
<given-names>Lanying</given-names>
</name>
<email>ldu@nybloodcenter.org</email>
<xref rid="aff0005" ref-type="aff">a</xref>
<xref rid="cor0005" ref-type="corresp">⁎</xref>
</contrib>
</contrib-group>
<aff id="aff0005"><label>a</label>
Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</aff>
<aff id="aff0010"><label>b</label>
Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease, University of Texas Medical Branch, Galveston, TX, USA</aff>
<aff id="aff0015"><label>c</label>
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA</aff>
<aff id="aff0020"><label>d</label>
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China</aff>
<aff id="aff0025"><label>e</label>
Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai, China</aff>
<author-notes><corresp id="cor0005"><label>⁎</label>
Corresponding authors at: New York Blood Center, Viral Immunology, 310 East 67th Street, New York, NY 10021, USA. Tel.: +1 212 570 3058/+1 212 570 3459; fax: +1 212 570 3099/+1 212 570 3299.. <email>sjiang@nybloodcenter.org</email>
<email>ldu@nybloodcenter.org</email>
</corresp>
<fn id="fn1"><label>1</label>
<p id="npar0005">These authors contributed equally to this work.</p>
</fn>
</author-notes>
<pub-date pub-type="pmc-release"><day>19</day>
<month>9</month>
<year>2014</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"><day>21</day>
<month>10</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="epub"><day>19</day>
<month>9</month>
<year>2014</year>
</pub-date>
<volume>32</volume>
<issue>46</issue>
<fpage>6170</fpage>
<lpage>6176</lpage>
<history><date date-type="received"><day>25</day>
<month>5</month>
<year>2014</year>
</date>
<date date-type="rev-recd"><day>20</day>
<month>7</month>
<year>2014</year>
</date>
<date date-type="accepted"><day>22</day>
<month>8</month>
<year>2014</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2014 Elsevier Ltd. All rights reserved.</copyright-statement>
<copyright-year>2014</copyright-year>
<copyright-holder>Elsevier Ltd</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 abstract-type="author-highlights" id="abs0005"><title>Highlights</title>
<p><list list-type="simple" id="lis0005"><list-item id="lsti0005"><label>•</label>
<p id="par0005">We express five MERS-CoV RBDs spanning S350-588, 358-588, 367-588, 377-606, and 377-588.</p>
</list-item>
<list-item id="lsti0010"><label>•</label>
<p id="par0010">S377-588, 367-588 and 358-588 have the highest receptor binding ability than others.</p>
</list-item>
<list-item id="lsti0015"><label>•</label>
<p id="par0015">S377-588 and 367-588 induced the highest-titer of IgG antibodies in mice.</p>
</list-item>
<list-item id="lsti0020"><label>•</label>
<p id="par0020">S377-588 elicits highest neutralization in mice and high neutralization in rabbits.</p>
</list-item>
<list-item id="lsti0025"><label>•</label>
<p id="par0025">S377-588 contains stably folded RBD structure and major neutralizing epitopes.</p>
</list-item>
</list>
</p>
</abstract>
<abstract id="abs0010"><p>The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is currently spreading among humans, making development of effective MERS vaccines a high priority. A defined receptor-binding domain (RBD) in MERS-CoV spike protein can potentially serve as a subunit vaccine candidate against MERS-CoV infections. To identify an ideal vaccine candidate, we have constructed five different versions of RBD fragments, S350-588-Fc, S358-588-Fc, S367-588-Fc, S367-606-Fc, and S377-588-Fc (their names indicate their residue range in the spike protein and their C-terminal Fc tag), and further investigated their receptor binding affinity, antigenicity, immunogenicity, and neutralizing potential. The results showed that S377-588-Fc is among the RBD fragments that demonstrated the highest DPP4-binding affinity and induced the highest-titer IgG antibodies in mice. In addition, S377-588-Fc elicited higher-titer neutralizing antibodies than all the other RBD fragments in mice, and also induced high-titer neutralizing antibodies in immunized rabbits. Structural analysis suggests that S377-588-Fc contains the stably folded RBD structure, the full receptor-binding site, and major neutralizing epitopes, such that additional structures to this fragment introduce non-neutralizing epitopes and may also alter the tertiary structure of the RBD. Taken together, our data suggest that the RBD fragment encompassing spike residues 377-588 is a critical neutralizing receptor-binding fragment and an ideal candidate for development of effective MERS vaccines, and that adding non-neutralizing structures to this RBD fragment diminishes its neutralizing potential. Therefore, in viral vaccine design, it is important to identify the most stable and neutralizing viral RBD fragment, while eliminating unnecessary and non-neutralizing structures, as a means of “immunofocusing”.</p>
</abstract>
<kwd-group id="kwd0005"><title>Keywords</title>
<kwd>MERS</kwd>
<kwd>MERS-CoV</kwd>
<kwd>Spike protein</kwd>
<kwd>Receptor-binding domain</kwd>
<kwd>Critical neutralizing domain</kwd>
<kwd>Immunofocusing</kwd>
</kwd-group>
</article-meta>
</front>
<body><sec id="sec0005"><label>1</label>
<title>Introduction</title>
<p id="par0030">An emerging infectious disease, Middle East respiratory syndrome (MERS) caused by MERS coronavirus (MERS-CoV), was first identified in 2012 in Saudi Arabia <xref rid="bib0005" ref-type="bibr">[1]</xref>
, and has since spread to other countries, including the United States. As of July 14, 2014, there have been 834 laboratory-confirmed cases, including 288 deaths, (<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/don/2014_07_14_mers/en/" id="intr0005">http://www.who.int/csr/don/2014_07_14_mers/en/</ext-link>
), raising serious concerns over its pandemic potential <xref rid="bib0010" ref-type="bibr">[2]</xref>
, <xref rid="bib0015" ref-type="bibr">[3]</xref>
. With bats and dromedary camels as its likely natural reservoir and intermediate transmission host, respectively <xref rid="bib0020" ref-type="bibr">[4]</xref>
, <xref rid="bib0025" ref-type="bibr">[5]</xref>
, <xref rid="bib0030" ref-type="bibr">[6]</xref>
, <xref rid="bib0035" ref-type="bibr">[7]</xref>
, <xref rid="bib0040" ref-type="bibr">[8]</xref>
, <xref rid="bib0045" ref-type="bibr">[9]</xref>
, <xref rid="bib0050" ref-type="bibr">[10]</xref>
, <xref rid="bib0055" ref-type="bibr">[11]</xref>
, MERS-CoV poses a long-term threat to human health <xref rid="bib0060" ref-type="bibr">[12]</xref>
, <xref rid="bib0065" ref-type="bibr">[13]</xref>
. Thus, the need for the development of effective prophylactic strategies, such as vaccines, to control the further spread of MERS-CoV is urgent.</p>
<p id="par0035">The spike (S) protein of MERS-CoV plays important roles in mediating viral entry to host cells <xref rid="bib0070" ref-type="bibr">[14]</xref>
. As the first step of cell entry, a defined receptor-binding domain (RBD) in the spike proteins binds to its functional receptor, dipeptidyl peptidase 4 (DPP4), on the host cell surface for viral attachment <xref rid="bib0075" ref-type="bibr">[15]</xref>
. Several versions of MERS-CoV RBD fragments have been identified by different groups. These RBD fragments encompass spike residues 358-588, 367-588, 377-588, and 367-606, respectively <xref rid="bib0080" ref-type="bibr">[16]</xref>
, <xref rid="bib0085" ref-type="bibr">[17]</xref>
, <xref rid="bib0090" ref-type="bibr">[18]</xref>
, <xref rid="bib0095" ref-type="bibr">[19]</xref>
, <xref rid="bib0100" ref-type="bibr">[20]</xref>
. Extensive studies have found that the spike RBD of SARS coronavirus (SARS-CoV), which caused the SARS epidemic in 2002–2003 <xref rid="bib0105" ref-type="bibr">[21]</xref>
, <xref rid="bib0110" ref-type="bibr">[22]</xref>
, is a critical neutralizing receptor-binding domain and an attractive subunit vaccine candidate against SARS-CoV infection <xref rid="bib0115" ref-type="bibr">[23]</xref>
, <xref rid="bib0120" ref-type="bibr">[24]</xref>
, <xref rid="bib0125" ref-type="bibr">[25]</xref>
, <xref rid="bib0130" ref-type="bibr">[26]</xref>
, <xref rid="bib0135" ref-type="bibr">[27]</xref>
, <xref rid="bib0140" ref-type="bibr">[28]</xref>
. It is likely that the MERS-CoV RBD could also serve as a subunit vaccine candidate against MERS-CoV infection. Indeed, it was previously shown that some of these MERS-CoV RBD fragments are immunogenic in animals, resulting in neutralizing antibody responses <xref rid="bib0085" ref-type="bibr">[17]</xref>
, <xref rid="bib0090" ref-type="bibr">[18]</xref>
. However, it is not clear which one of these RBD fragments represents an ideal vaccine candidate and what is the mechanism behind the potential differences in the neutralizing abilities of these RBD fragments.</p>
<p id="par0040">In this study, we have expressed each of these MERS-CoV RBD fragments that were fused with Fc fragment of human IgG, and investigated their receptor binding affinity, antigenicity, immunogenicity, and neutralizing potential. We have found the RBD fragment with the most neutralizing potential, and explained the mechanism behind it. Overall, this study has identified an ideal vaccine candidate for controlling MERS-CoV infections, and enhanced understanding of design strategies for vial subunit vaccines.</p>
</sec>
<sec id="sec0010"><label>2</label>
<title>Materials and methods</title>
<sec id="sec0045"><label>2.1</label>
<title>Ethics statement</title>
<p id="par0045">Four- to six-week-old female BALB/c mice and four- to five-month-old female NZW rabbits were used in the study. The animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of the New York Blood Center (Permit Number: 194.15).</p>
</sec>
<sec id="sec0050"><label>2.2</label>
<title>Construction, expression and purification of recombinant proteins</title>
<p id="par0050">The construction, expression and purification of recombinant MERS-CoV RBD fragments with Fc of human IgG were done as previously described with some modifications <xref rid="bib0090" ref-type="bibr">[18]</xref>
, <xref rid="bib0145" ref-type="bibr">[29]</xref>
. Briefly, genes encoding residues 350-588, 358-588, 367-588, 367-606, and 377-588 of MERS-CoV S protein were respectively amplified by PCR using codon-optimized MERS-CoV S sequences (GenBank: <ext-link ext-link-type="uri" xlink:href="ncbi-p:AFS88936.1" id="intr0010">AFS88936.1</ext-link>
) as template and inserted into the pFUSE-hIgG1-Fc2 expression vector (hereinafter named Fc; InvivoGen, San Diego, CA). MERS-CoV S1 (residues 18-725) plus a C-terminal His<sub>6</sub>
(S1-His) was amplified and inserted into pJW4303 expression vector (Jiangsu Taizhou Haiyuan Protein Biotech, Co., Ltd., China). The recombinant plasmids were transfected into 293T cells (ATCC, Manassas, VA), changed into fresh serum-free DMEM (Invitrogen, Carlsbad, CA) 8 h later, and collected for supernatant containing expressed proteins 72 h post-transfection. The recombinant proteins were purified by Protein A affinity chromatography (GE Healthcare, Piscataway, NJ) for proteins with Fc or Ni-NTA Superflow (Qiagen, Valencia, CA) for proteins with His tag, according to the manufacturers’ instructions.</p>
<p id="par0055">Human DPP4 ectodomain (residues 39-766) was expressed and purified as previously described <xref rid="bib0095" ref-type="bibr">[19]</xref>
. Briefly, recombinant human DPP4 ectodomain containing an N-terminal honeybee melittin signal peptide and a C-terminal His<sub>6</sub>
was expressed in insect cells using the Bac-to-Bac expression system (Invitrogen), secreted into cell culture medium, and subsequently purified by Ni-NTA affinity column and Superdex200 gel filtration column (GE Healthcare).</p>
</sec>
<sec id="sec0055"><label>2.3</label>
<title>SDS-PAGE and Western blot</title>
<p id="par0060">The purified MERS-CoV RBD fragments were analyzed by SDS-PAGE and Western blot as previously described <xref rid="bib0145" ref-type="bibr">[29]</xref>
, <xref rid="bib0150" ref-type="bibr">[30]</xref>
. Briefly, the boiled and nonboiled proteins were separated by 10% Tris–Glycine SDS-PAGE gels, followed by transferring to nitrocellulose membranes. After blocking overnight at 4 °C using 5% non-fat milk in PBST, the blots were incubated for 1 h at room temperature with MERS-CoV S-specific polyclonal antibodies (1:1000). After three washes, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:3000, Invitrogen) for 1 h at room temperature, and the signals were visualized using ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).</p>
</sec>
<sec id="sec0060"><label>2.4</label>
<title>Co-immunoprecipitation assay</title>
<p id="par0065">The binding of MERS-CoV RBD fragments with DPP4 was performed by co-immunoprecipitation assay as previously described <xref rid="bib0090" ref-type="bibr">[18]</xref>
. Briefly, recombinant soluble DPP4 (sDPP4) (10 μg) or DPP4-expressing Huh-7 cell lysates (5 × 10<sup>7</sup>
/ml) were respectively incubated with MERS-CoV RBD fragments (10 μg) plus Protein A Sepharose Beads at 4 °C for 1 h, followed by washing with lysis buffer and PBS, and boiling for 10 min. The samples were subjected to SDS-PAGE and Western blot analysis, followed by detection using anti-DPP4 monoclonal antibody (1 μg/ml, R&D Systems, Minneapolis, MN) and MERS-CoV S1-specific polyclonal antibodies (1:1000), respectively.</p>
</sec>
<sec id="sec0065"><label>2.5</label>
<title>Animal vaccination and sample collection</title>
<p id="par0070">Mice were immunized with MERS-CoV RBD fragments following a previously described protocol <xref rid="bib0090" ref-type="bibr">[18]</xref>
, <xref rid="bib0145" ref-type="bibr">[29]</xref>
. Briefly, mice were prime-vaccinated (subcutaneously, s.c.) with 10 μg/mouse of recombinant MERS-CoV RBD fragments in the presence of Montanide ISA 51 adjuvant (SEPPIC, Fairfield, NJ), and boosted twice with the same immunogen and adjuvant at 3-week (21 days) intervals. Sera from 10 days post-last vaccination (52 days) were heat-inactivated at 56 °C for 30 min, and detected for MERS-CoV S-specific antibody response and neutralizing antibodies. Sera from 0, 10, 31, and 136 days post-initial immunization of S377-588-Fc were also tested for MERS-CoV S-specific antibody response.</p>
<p id="par0075">The rabbit immunization (Covance Research Products Inc., Denver, PA) followed a protocol similar to that of mice with some modifications. Briefly, rabbits were prime-vaccinated with MERS-CoV S377-588-Fc fragment (250 μg/rabbit) plus Freund's complete adjuvant and then boosted twice at 3-week intervals with the same immunogen (125 μg/rabbit) plus Freund's incomplete adjuvant. Sera were collected and tested as above.</p>
</sec>
<sec id="sec0070"><label>2.6</label>
<title>ELISA</title>
<p id="par0080">ELISA was carried out to test the binding of MERS-CoV RBD fragments to sDPP4 as previously described <xref rid="bib0090" ref-type="bibr">[18]</xref>
. Briefly, 96-well ELISA plates were precoated with sDPP4 (4 μg/ml) overnight at 4 °C and blocked with 2% non-fat milk at 37 °C for 2 h. Diluted MERS-CoV RBD fragments were added to the plates and incubated at 37 °C for 1 h, followed by four washes. Bound antibodies were incubated with HRP-conjugated anti-human IgG (1:3000, Invitrogen) at 37 °C for 1 h. The reaction was visualized by substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Invitrogen) and stopped by 1 N H<sub>2</sub>
SO<sub>4</sub>
. The absorbance at 450 nm (A450) was measured by ELISA plate reader (Tecan, San Jose, CA).</p>
<p id="par0085">The detection of MERS-CoV S-specific antibody responses in collected mouse sera was performed following a protocol similar to that described above, except for replacing the coating antigen with MERS-CoV S1-His protein (1 μg/ml), the primary antibody with serially diluted mouse sera, and the secondary antibody with anti-mouse IgG-HRP (1:3000, Invitrogen) <xref rid="bib0155" ref-type="bibr">[31]</xref>
.</p>
</sec>
<sec id="sec0075"><label>2.7</label>
<title>Neutralization assay</title>
<p id="par0090">The standard micro-neutralization assay was used to quantify the neutralizing potential of each of the RBD fragments, as previously described <xref rid="bib0090" ref-type="bibr">[18]</xref>
, <xref rid="bib0155" ref-type="bibr">[31]</xref>
, <xref rid="bib0160" ref-type="bibr">[32]</xref>
. Briefly, serum samples were diluted at serial 2-fold in 96-well tissue culture plates, and incubated for 1 h at room temperature with ∼100 infectious MERS-CoV/EMC-2012/well before transferring to duplicate wells of Vero E6 cells grown in 96-well tissue culture plates. After 72 h of incubation, when the virus control wells exhibited advanced virus-induced cytopathic effect (CPE), the neutralizing capacity of individual serum samples was assessed by determining the presence or absence of CPE. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited virus-induced CPE in at least 50% of the wells (NT<sub>50</sub>
).</p>
</sec>
<sec id="sec0080"><label>2.8</label>
<title>Statistical analysis</title>
<p id="par0095">Values are presented as mean with standard deviation (SD). Statistical significance among different vaccination groups was calculated by Student's <italic>t</italic>
-test using Stata statistical software. <italic>P</italic>
values less than 0.05 were considered statistically significant.</p>
</sec>
</sec>
<sec id="sec0015"><label>3</label>
<title>Results</title>
<sec id="sec0020"><label>3.1</label>
<title>Characterization of MERS-CoV RBD protein fragments</title>
<p id="par0100">Five human IgG Fc-fused fragments representing the defined RBD residues 358-588, 367-588, 367-606, and 377-588 of MERS-CoV spike protein and the RBD residues 350-588 control were constructed, expressed, and subjected to antigenicity measurement, using the procedures as previously described (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>
B) <xref rid="bib0090" ref-type="bibr">[18]</xref>
. All five RBD fragments were expressed at high levels from culture supernatants of transfected 293 T cells and purified to high homogeneity. The C-terminal Fc tag strongly promoted formation of RBD dimers, as evidenced by the almost twice of the molecular weight of non-denatured <italic>versus</italic>
denatured proteins (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>
C, top). These recombinant RBD fragments reacted strongly with polyclonal antibodies specifically raised in mice against recombinant S1 protein of MERS-CoV (<xref rid="fig0005" ref-type="fig">Fig. 1</xref>
C, bottom), suggesting that they are in their native and antigenic conformation.<fig id="fig0005"><label>Fig. 1</label>
<caption><p><bold>Construction and characterization of MERS-CoV RBD protein fragments.</bold>
(A) Schematic structure of MERS-CoV S1 subunit. SP, signal peptide. (B) Constructed plasmids encoding MERS-CoV RBD fragments fused with human IgG Fc. IL2ss: IL2 signal sequence that directs the secretion of the expressed protein into the culture supernatant. (C) SDS-PAGE and Western blot analysis of the purified RBD fragments. Denatured (boiled) or non-denatured (nonboiled) samples (5 μg) were subjected to SDS-PAGE (top) and Western blot analysis (bottom) using anti-MERS-CoV S1 antibodies. The molecular weight marker (kDa) is indicated on the left.</p>
</caption>
<graphic xlink:href="gr1"></graphic>
</fig>
</p>
</sec>
<sec id="sec0025"><label>3.2</label>
<title>Receptor binding affinity of MERS-CoV RBD fragments</title>
<p id="par0105">Two alternative assays, co-immunoprecipitation and ELISA, were carried out to measure the receptor-binding activities of MERS-CoV RBD fragments. Results from co-immunoprecipitation assay showed that the five MERS-CoV RBD fragments were co-immunoprecipitated with recombinant sDPP4 protein. Two clear bands corresponding to the sizes of DPP4 and respective Fc-fused MERS-CoV RBD fragments were readily revealed with anti-DPP4- and anti-MERS-CoV S1-specific antibodies, whereas a single band with a molecular weight corresponding to DPP4 was detected in the sample containing only sDPP4 (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>
A, left). In addition, each of the five MERS-CoV RBD fragments co-immunoprecipitated cell-associated DPP4 anchored on Huh-7 cells, in which target proteins with anticipated sizes were identified using anti-DPP4- and anti-MERS-CoV S1-specific antibodies, respectively (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>
A, right). Thus, co-immunoprecipitation assay demonstrated that all of the five RBD fragments specifically interact with MERS-CoV's receptor DPP4.<fig id="fig0010"><label>Fig. 2</label>
<caption><p><bold>Receptor-binding affinity of MERS-CoV RBD fragments.</bold>
(A) Co-immunoprecipitation assays, followed by Western blot analyses, were performed to determine the binding affinity between the RBD fragments and DPP4 receptor. Briefly, purified recombinant RBD fragments were incubated with either 10 μg sDPP4 (left) or DPP4-expressing Huh-7 cells (right) in the presence of Protein A Sepharose beads before subjecting to Western blot analysis with either anti-DPP4 (1 μg/ml, top) or anti-MERS-CoV S1 antibodies (1:1000, bottom). (B) ELISA was carried out to measure the binding affinity between the RBD fragments and recombinant sDPP4. Recombinant human IgG Fc (hIgG-Fc) protein was included as the negative control. There were significant differences between three of the RBD fragments (S358-588-Fc, S367-588-Fc, and S377-588-Fc) and the other two (S350-588-Fc and S367-606-Fc) both at 50 (*) and 12.5 (#) μg/ml, respectively.</p>
</caption>
<graphic xlink:href="gr2"></graphic>
</fig>
</p>
<p id="par0110">ELISA was then performed by adding MERS-CoV RBD fragments to the plates coated with a fixed amount of sDPP4, followed by identification of the binding affinity based on the OD values at 450 nm. While all of the tested MERS-CoV RBDs were capable of binding to sDPP4, we noted the existence of their differential binding capacity, as shown in <xref rid="fig0010" ref-type="fig">Fig. 2</xref>
B. Among the five RBD fragments, S358-588-Fc, S367-588-Fc and S377-588-Fc possessed significantly higher DPP4-binding affinity than S350-588-Fc and S367-606-Fc at concentrations of 50 and 12.5 μg/ml, respectively. As expected, the control hIgG-Fc protein did not show any specific binding to sDPP4 (<xref rid="fig0010" ref-type="fig">Fig. 2</xref>
B). Therefore, the extended N- or C-terminus of MERS-CoV RBD fragments appears to have negative effect on the receptor-binding activity.</p>
</sec>
<sec id="sec0030"><label>3.3</label>
<title>Immunogenicity of MERS-CoV RBD fragments</title>
<p id="par0115">To evaluate the immunogenicity of the five MERS-CoV RBD fragments, we immunized mice with each of the RBD fragments according to the dosing strategy described in Materials and Methods, and detected specific responses of total IgG antibody, along with IgG1 and IgG2a subtypes, in serum specimens of immunized mice. ELISA results showed that all five RBD fragments were immunogenic in mice, and induced production of IgG antibodies that specifically bind to MERS-CoV S1 protein without the fusion of Fc. Particularly, sera from mice immunized with S367-588-Fc or S377-588-Fc acquired a stronger binding to MERS-CoV S1 protein than those immunized with the other three RBD fragments, whereas the control samples from PBS-immunized mice revealed negligible binding activity (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>
A), confirming the specificity of such IgG antibody response elicited by these RBD fragments. In addition, the endpoint titration of IgG antibody response revealed that S367-588-Fc and S377-588-Fc were more immunogenic than S350-588-Fc, S358-588-Fc or S367-606-Fc in eliciting MERS-CoV S1-specific IgG antibodies in mice after 3 vaccinations (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>
B). Thus, the extended N- or C-terminus of the MERS-CoV RBD also reduces the immunogenicity of the RBD fragments.<fig id="fig0015"><label>Fig. 3</label>
<caption><p><bold>Immunogenicity of MERS-CoV RBD fragments.</bold>
Sera collected at 10 days post-last immunization with each of the RBD fragments were the subjects to immunogenicity study, with the serum specimens of sham/PBS-immunized mice included as the negative control. The data are presented as mean ±SD from five mice in each group. (A) Total IgG antibodies specific to MERS-CoV S1 protein were assessed by ELISA. (B) MERS-CoV S1-specific IgG antibodies elicited by groups of differentially immunized mice were determined by ELISA, and the titers were expressed as the endpoint dilutions that remain positively detectable. There were significant differences between two of the RBD fragments (S367-588-Fc and S377-588-Fc) and the other three (S350-588-Fc, S358-588-Fc, and S367-606-Fc). (C) MERS-CoV S1-specific IgG1 subtype antibody titers of groups of mice immunized with indicated RBD fragments were compared, based on the endpoint analysis by ELISA. There were significant differences between three of the RBD fragments (S350-588-Fc, S367-588-Fc, and S377-588-Fc), and S367-606-Fc (*), and between two of the RBD fragments (S350-588-Fc and S367-588-Fc) and S358-588-Fc (#). (D) The endpoint titers of MERS-CoV S1-specific IgG2a subtype antibodies derived from mice immunized with different RBD fragments were compared. There were significant differences between four of the RBD fragments (S350-588-Fc, S358-588-Fc, S367-588-Fc, and S377-588-Fc) and S367-606-Fc (*), or between S367-588-Fc and three of the RBD fragments (S350-588-Fc (#), S358-588-Fc (■), and S377-588-Fc (●)), respectively.</p>
</caption>
<graphic xlink:href="gr3"></graphic>
</fig>
</p>
<p id="par0120">Observation of IgG subtypes revealed that S350-588-Fc, S367-588-Fc and S377-588-Fc were capable of inducing a significantly higher level of IgG1 (Th2) antibody response than S367-606-Fc, while S350-588-Fc and S367-588-Fc elicited IgG1 antibody significantly higher than S358-588-Fc (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>
C). In addition, S350-588-Fc, S358-588-Fc, S367-588-Fc and S377-588-Fc promoted a significantly higher level of IgG2a (Th1) antibody response than S367-606-Fc, whereas the IgG2a antibodies induced by S367-588-Fc were also significantly higher than those induced by S350-588-Fc, S358-588-Fc and S377-588-Fc, respectively (<xref rid="fig0015" ref-type="fig">Fig. 3</xref>
D). The above results suggest that S377-588-Fc is among the RBD fragments that induced high titers of Th1 and Th2 antibody response.</p>
</sec>
<sec id="sec0035"><label>3.4</label>
<title>S377-588-Fc having the best potential to induce highly potent neutralizing antibody responses in vaccinated animals</title>
<p id="par0125">Neutralizing potentials of the five RBD fragments were initially evaluated in immunized mice. Results from Vero E6-based micro-neutralization showed that each of the five RBD fragments was capable of inducing neutralization antibodies. Among them, S377-588-Fc induced the highest level of neutralizing antibody response on the 10th day after the third immunization (<xref rid="fig0020" ref-type="fig">Fig. 4</xref>
A). As expected, sera from PBS-immunized mice did not show neutralizing activity. Thus, S377-588-Fc has the highest neutralizing potential among the five RBD fragments. Furthermore, S377-588-Fc was used to immunize rabbits to confirm its neutralizing potential in different animal species. Vero E6-based micro-neutralization assay demonstrated that on the 10th day after the third immunization with S377-588-Fc, high titers of neutralizing antibody responses were generated in rabbits (<xref rid="fig0020" ref-type="fig">Fig. 4</xref>
B). Therefore, S377-588-Fc, the shortest among the five RBD fragments, can produce high-titer neutralizing antibody responses in both mice and rabbits, suggesting that the fragment containing residues 377-588 of MERS-CoV spike protein is a critical neutralizing receptor-binding domain.<fig id="fig0020"><label>Fig. 4</label>
<caption><p><bold>Neutralization potentials of MERS-CoV RBD fragments.</bold>
The aforementioned serum specimens were subjected to Vero E6-based micro-neutralization assays against live MERS-CoV infection. Neutralizing antibody titers are expressed as the reciprocal of the highest dilutions of specimens tested that resulted in a complete inhibition of virus-induced CPE in at least 50% of the wells (NT<sub>50</sub>
). (A) Neutralizing antibody titers in sera of mice immunized with MERS-CoV RBD fragments. Sera from 10 days post-3rd immunization were used for the test and the data are presented as mean ± SD from five mice in each group. There were significant differences between S377-588-Fc and the other four RBD fragments. (B) Neutralizing antibody titers in sera of rabbits (R1 and R2) immunized with S377-588-Fc protein. The sera from pre-immunization and 10 days post-3rd immunization were used for the test. The data are presented as mean ± SD from duplicate wells. (C) The endpoint titers of MERS-CoV S1-specific IgG antibodies by ELISA in S377-588-Fc-immunized mouse sera at 0, 10, 31, 52, and 136 days post-immunization. The data are presented as mean ± SD from five mice in each group.</p>
</caption>
<graphic xlink:href="gr4"></graphic>
</fig>
</p>
<p id="par0130">To elucidate the potential of S377-588-Fc as a vaccine candidate, we immunized mice with this protein for a period of 136 days, and observed the kinetics of antibody responses. As expected, increasing titers of IgG antibodies were found in mouse sera after initial immunization, and reached the highest titers during 31 and 52 days post-immunization. The antibody titers decreased slightly afterwards, but still maintained at relative high levels (<xref rid="fig0020" ref-type="fig">Fig. 4</xref>
C). These data demonstrated that the fragment containing residues 377-588 of MERS-CoV spike protein is able to induce and maintain RBD-specific immune responses in the vaccinated animals for at least four months, confirming its good potential to be further developed as a subunit MERS vaccine.</p>
</sec>
</sec>
<sec id="sec0040"><label>4</label>
<title>Discussion</title>
<p id="par0135">The increasing number of MERS cases indicates urgency for the development of effective vaccines against MERS-CoV infection and disease. Among all of the MERS-CoV structural proteins, the spike protein, particularly its RBD, is an important target for such an effort, due to its proven ability to elicit potent neutralizing antibody response in immunized laboratory animals <xref rid="bib0165" ref-type="bibr">[33]</xref>
. Indeed, recombinant RBD fragments containing residues 358-588 and 377-662 of MERS-CoV S have been shown to induce neutralizing antibody response in immunized rabbits and mice, respectively <xref rid="bib0085" ref-type="bibr">[17]</xref>
, <xref rid="bib0145" ref-type="bibr">[29]</xref>
. Moreover, a modified vaccinia virus Ankara (MVA) expressing the full-length S protein of MERS-CoV was also proven effective in vaccinated mice to produce potent neutralizing antibodies and restricted MERS-CoV infection <italic>in vivo</italic>
<xref rid="bib0170" ref-type="bibr">[34]</xref>
. In addition to the aforementioned recombinant proteins and viral vectors, nanoparticles and Venezuelan Equine Encephalitis Virus Replicon Particles (VRPs)-expressing spike protein of MERS-CoV have also shown great potentials as MERS vaccines <xref rid="bib0175" ref-type="bibr">[35]</xref>
, <xref rid="bib0180" ref-type="bibr">[36]</xref>
.</p>
<p id="par0140">In this study, we expressed five different versions of MERS-CoV RBD fragments based on the previously published studies <xref rid="bib0080" ref-type="bibr">[16]</xref>
, <xref rid="bib0085" ref-type="bibr">[17]</xref>
, <xref rid="bib0090" ref-type="bibr">[18]</xref>
, <xref rid="bib0095" ref-type="bibr">[19]</xref>
, and further investigated and compared their receptor binding ability, antigenicity, immunogenicity, and neutralizing potential. The Fc of human IgG was fused to these RBD fragments because the Fc tag can enhance the expression, purification, and immunogenicity of viral RBD proteins <xref rid="bib0185" ref-type="bibr">[37]</xref>
, <xref rid="bib0190" ref-type="bibr">[38]</xref>
, <xref rid="bib0195" ref-type="bibr">[39]</xref>
, <xref rid="bib0200" ref-type="bibr">[40]</xref>
, <xref rid="bib0205" ref-type="bibr">[41]</xref>
, <xref rid="bib0210" ref-type="bibr">[42]</xref>
. Our results show that the abilities of these RBD fragments to induce immune responses and neutralizing antibodies differ significantly. Among these RBD fragments, S377-588-Fc demonstrated high receptor-binding affinity, induced high titer IgG antibody in mice, elicited the highest titer neutralizing antibodies in mice, and also induced high titer neutralizing antibodies in rabbits.</p>
<p id="par0145">Crystal structure of MERS-CoV RBD reveals that RBD fragment encompassing residues 377-588 of MERS-CoV spike protein has a stably folded structure with ordered residues on the N- and C-termini. It contains the receptor-binding motif (RBM, residues 484-567) (<xref rid="fig0025" ref-type="fig">Fig. 5</xref>
) and almost all of the major neutralizing epitopes identified in MERS-CoV RBD, many of which are specifically recognized by neutralizing monoclonal antibodies (nAbs) <xref rid="bib0155" ref-type="bibr">[31]</xref>
, <xref rid="bib0215" ref-type="bibr">[43]</xref>
, <xref rid="bib0220" ref-type="bibr">[44]</xref>
, <xref rid="bib0225" ref-type="bibr">[45]</xref>
. These epitopes are clustered on a protruding ridge in the RBM region of the RBD, and overlap with the DPP4-binding site (<xref rid="fig0025" ref-type="fig">Fig. 5</xref>
) <xref rid="bib0080" ref-type="bibr">[16]</xref>
, <xref rid="bib0095" ref-type="bibr">[19]</xref>
, <xref rid="bib0100" ref-type="bibr">[20]</xref>
. Although not yet experimentally confirmed, other regions in the RBM that overlap with the DPP4-binding site may also contain neutralizing epitopes. On the other hand, the regions in the MERS-CoV spike that do not overlap with the DPP4-binding site likely contain non-neutralizing epitopes. These non-neutralizing epitopes might hinder the production of neutralizing antibodies and may also induce harmful immune responses <xref rid="bib0230" ref-type="bibr">[46]</xref>
, <xref rid="bib0235" ref-type="bibr">[47]</xref>
, <xref rid="bib0240" ref-type="bibr">[48]</xref>
. The regions in the RBD core structures contribute to the folding and the stability of the whole RBD, and thus they are essential. However, the regions flanking the RBD do not contribute to protein folding or receptor binding. Therefore, it is necessary to identify the critical neutralizing epitopes in RBD in the MERS-CoV spike protein, and exclude non-neutralizing epitopes, without the expense of domain integrity and stability, as a means of “immunofocusing” for vaccine development.<fig id="fig0025"><label>Fig. 5</label>
<caption><p><bold>Crystal structure of MERS-CoV spike RBD (PDB 4L3N)</bold>
<xref rid="bib0095" ref-type="bibr">[19]</xref>
. MERS-CoV spike RBD is a well-folded structure, and contains a core structure (in blue) and a receptor-binding motif (RBM; in magenta). Helices are drawn as cylinders, and strands as arrows. The crystallized RBD fragment encompasses residues 367 to 588, with the N- and C-terminus labeled as N* and C*, respectively. The N-terminal region of the RBD fragment (from residues 369 to 378) is disordered, as indicated by a dashed line. Neutralizing epitopes recognized by known nAbs are indicated in RBM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</p>
</caption>
<graphic xlink:href="gr5"></graphic>
</fig>
</p>
<p id="par0150">It is important to further test the immune efficacy of S377-588-Fc in an animal model challenged by MERS-CoV. Non-human primates, such as rhesus macaques, can be infected by MERS-CoV, showing clinical signs of the disease and histopathological changes in lungs <xref rid="bib0245" ref-type="bibr">[49]</xref>
, <xref rid="bib0250" ref-type="bibr">[50]</xref>
. However, the high cost of purchasing and maintaining the monkeys and performing the experiments in ABSL-3 facility have prevented us from using this animal model. Small animals such as mice, Syrian hamsters, and ferrets that can be infected by SARS-CoV are unsusceptible to MERS-CoV infection <xref rid="bib0255" ref-type="bibr">[51]</xref>
, <xref rid="bib0260" ref-type="bibr">[52]</xref>
, <xref rid="bib0265" ref-type="bibr">[53]</xref>
, <xref rid="bib0270" ref-type="bibr">[54]</xref>
. Although mice transduced with adenoviral vectors expressing human DPP4 can be infected by MERS-CoV, they did not show significant clinical symptoms of MERS <xref rid="bib0275" ref-type="bibr">[55]</xref>
. Therefore, the <italic>in vivo</italic>
efficacy of this MERS-CoV RBD-based vaccine candidate will be evaluated in a more sophisticated small animal model once it becomes available.</p>
<p id="par0155">Overall, the RBD fragment encompassing residues 377-588 of MERS-CoV S protein is the ideal subunit vaccine candidate with the capability of inducing strong neutralizing antibodies in different species of animals. Therefore, it can be the focus of future development of MERS vaccines. Furthermore, this study suggests that in vaccine design it maybe helpful to eliminate non-neutralizing epitopes from the RBD fragments to keep the RBD fragments in their most physiological relevant conformation, and also allow the host immune responses to focus on the neutralizing epitopes.</p>
</sec>
<sec id="sec0085"><title>Conflict of interest statement</title>
<p id="par0170">The authors declared no conflict of interest.</p>
</sec>
</body>
<back><ref-list id="bibl0005"><title>References</title>
<ref id="bib0005"><label>1</label>
<element-citation publication-type="journal" id="sbref0005"><person-group person-group-type="author"><name><surname>Zaki</surname>
<given-names>A.M.</given-names>
</name>
<name><surname>van</surname>
<given-names>B.S.</given-names>
</name>
<name><surname>Bestebroer</surname>
<given-names>T.M.</given-names>
</name>
<name><surname>Osterhaus</surname>
<given-names>A.D.</given-names>
</name>
<name><surname>Fouchier</surname>
<given-names>R.A.</given-names>
</name>
</person-group>
<article-title>Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia</article-title>
<source>N Engl J 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="bib0010"><label>2</label>
<element-citation publication-type="journal" id="sbref0010"><person-group person-group-type="author"><name><surname>Gulland</surname>
<given-names>A.</given-names>
</name>
</person-group>
<article-title>WHO voices concern over rising numbers of MERS-CoV cases</article-title>
<source>BMJ</source>
<volume>348</volume>
<year>2014</year>
<fpage>g2968</fpage>
<pub-id pub-id-type="pmid">24778282</pub-id>
</element-citation>
</ref>
<ref id="bib0015"><label>3</label>
<element-citation publication-type="journal" id="sbref0015"><person-group person-group-type="author"><name><surname>Kupferschmidt</surname>
<given-names>K.</given-names>
</name>
</person-group>
<article-title>Emerging diseases. Soaring MERS cases in Saudi Arabia raise alarms</article-title>
<source>Science</source>
<volume>344</volume>
<year>2014</year>
<fpage>457</fpage>
<lpage>458</lpage>
<pub-id pub-id-type="pmid">24786052</pub-id>
</element-citation>
</ref>
<ref id="bib0020"><label>4</label>
<element-citation publication-type="journal" id="sbref0020"><person-group person-group-type="author"><name><surname>Memish</surname>
<given-names>Z.A.</given-names>
</name>
<name><surname>Mishra</surname>
<given-names>N.</given-names>
</name>
<name><surname>Olival</surname>
<given-names>K.J.</given-names>
</name>
<name><surname>Fagbo</surname>
<given-names>S.F.</given-names>
</name>
<name><surname>Kapoor</surname>
<given-names>V.</given-names>
</name>
<name><surname>Epstein</surname>
<given-names>J.H.</given-names>
</name>
</person-group>
<article-title>Middle East respiratory syndrome coronavirus in bats, Saudi Arabia</article-title>
<source>Emerg Infect Dis</source>
<volume>19</volume>
<year>2013</year>
<fpage>1819</fpage>
<lpage>1823</lpage>
<pub-id pub-id-type="pmid">24206838</pub-id>
</element-citation>
</ref>
<ref id="bib0025"><label>5</label>
<element-citation publication-type="journal" id="sbref0025"><person-group person-group-type="author"><name><surname>Perera</surname>
<given-names>R.A.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>P.</given-names>
</name>
<name><surname>Gomaa</surname>
<given-names>M.R.</given-names>
</name>
<name><surname>El-Shesheny</surname>
<given-names>R.</given-names>
</name>
<name><surname>Kandeil</surname>
<given-names>A.</given-names>
</name>
<name><surname>Bagato</surname>
<given-names>O.</given-names>
</name>
</person-group>
<article-title>Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013</article-title>
<source>Euro Surveill</source>
<volume>18</volume>
<year>2013</year>
<comment>pii:20574</comment>
</element-citation>
</ref>
<ref id="bib0030"><label>6</label>
<element-citation publication-type="journal" id="sbref0030"><person-group person-group-type="author"><name><surname>Reusken</surname>
<given-names>C.B.</given-names>
</name>
<name><surname>Haagmans</surname>
<given-names>B.L.</given-names>
</name>
<name><surname>Muller</surname>
<given-names>M.A.</given-names>
</name>
<name><surname>Gutierrez</surname>
<given-names>C.</given-names>
</name>
<name><surname>Godeke</surname>
<given-names>G.J.</given-names>
</name>
<name><surname>Meyer</surname>
<given-names>B.</given-names>
</name>
</person-group>
<article-title>Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study</article-title>
<source>Lancet Infect Dis</source>
<volume>13</volume>
<year>2013</year>
<fpage>859</fpage>
<lpage>866</lpage>
<pub-id pub-id-type="pmid">23933067</pub-id>
</element-citation>
</ref>
<ref id="bib0035"><label>7</label>
<element-citation publication-type="journal" id="sbref0035"><person-group person-group-type="author"><name><surname>Azhar</surname>
<given-names>E.I.</given-names>
</name>
<name><surname>El-Kafrawy</surname>
<given-names>S.A.</given-names>
</name>
<name><surname>Farraj</surname>
<given-names>S.A.</given-names>
</name>
<name><surname>Hassan</surname>
<given-names>A.M.</given-names>
</name>
<name><surname>Al-Saeed</surname>
<given-names>M.S.</given-names>
</name>
<name><surname>Hashem</surname>
<given-names>A.M.</given-names>
</name>
</person-group>
<article-title>Evidence for camel-to-human transmission of MERS coronavirus</article-title>
<source>N Engl J Med</source>
<volume>370</volume>
<year>2014</year>
<fpage>2499</fpage>
<lpage>2505</lpage>
<pub-id pub-id-type="pmid">24896817</pub-id>
</element-citation>
</ref>
<ref id="bib0040"><label>8</label>
<element-citation publication-type="journal" id="sbref0040"><person-group person-group-type="author"><name><surname>Chu</surname>
<given-names>D.K.</given-names>
</name>
<name><surname>Poon</surname>
<given-names>L.L.</given-names>
</name>
<name><surname>Gomaa</surname>
<given-names>M.M.</given-names>
</name>
<name><surname>Shehata</surname>
<given-names>M.M.</given-names>
</name>
<name><surname>Perera</surname>
<given-names>R.A.</given-names>
</name>
<name><surname>Abu</surname>
<given-names>Z.D.</given-names>
</name>
</person-group>
<article-title>MERS coronaviruses in dromedary camels, Egypt</article-title>
<source>Emerg Infect Dis</source>
<volume>20</volume>
<year>2014</year>
<fpage>1049</fpage>
<lpage>1053</lpage>
<pub-id pub-id-type="pmid">24856660</pub-id>
</element-citation>
</ref>
<ref id="bib0045"><label>9</label>
<element-citation publication-type="journal" id="sbref0045"><person-group person-group-type="author"><name><surname>Meyer</surname>
<given-names>B.</given-names>
</name>
<name><surname>Muller</surname>
<given-names>M.A.</given-names>
</name>
<name><surname>Corman</surname>
<given-names>V.M.</given-names>
</name>
<name><surname>Reusken</surname>
<given-names>C.B.</given-names>
</name>
<name><surname>Ritz</surname>
<given-names>D.</given-names>
</name>
<name><surname>Godeke</surname>
<given-names>G.J.</given-names>
</name>
</person-group>
<article-title>Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013</article-title>
<source>Emerg Infect Dis</source>
<volume>20</volume>
<year>2014</year>
<fpage>552</fpage>
<lpage>559</lpage>
<pub-id pub-id-type="pmid">24655412</pub-id>
</element-citation>
</ref>
<ref id="bib0050"><label>10</label>
<element-citation publication-type="journal" id="sbref0050"><person-group person-group-type="author"><name><surname>Briese</surname>
<given-names>T.</given-names>
</name>
<name><surname>Mishra</surname>
<given-names>N.</given-names>
</name>
<name><surname>Jain</surname>
<given-names>K.</given-names>
</name>
<name><surname>Zalmout</surname>
<given-names>I.S.</given-names>
</name>
<name><surname>Jabado</surname>
<given-names>O.J.</given-names>
</name>
<name><surname>Karesh</surname>
<given-names>W.B.</given-names>
</name>
</person-group>
<article-title>Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in Saudi Arabia</article-title>
<source>mBio</source>
<volume>5</volume>
<year>2014</year>
<fpage>e01146</fpage>
<lpage>e1214</lpage>
<pub-id pub-id-type="pmid">24781747</pub-id>
</element-citation>
</ref>
<ref id="bib0055"><label>11</label>
<element-citation publication-type="journal" id="sbref0055"><person-group person-group-type="author"><name><surname>Alagaili</surname>
<given-names>A.N.</given-names>
</name>
<name><surname>Briese</surname>
<given-names>T.</given-names>
</name>
<name><surname>Mishra</surname>
<given-names>N.</given-names>
</name>
<name><surname>Kapoor</surname>
<given-names>V.</given-names>
</name>
<name><surname>Sameroff</surname>
<given-names>S.C.</given-names>
</name>
<name><surname>Burbelo</surname>
<given-names>P.D.</given-names>
</name>
</person-group>
<article-title>Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia</article-title>
<source>mBio</source>
<volume>5</volume>
<year>2014</year>
<fpage>e00884</fpage>
<lpage>e914</lpage>
<pub-id pub-id-type="pmid">24570370</pub-id>
</element-citation>
</ref>
<ref id="bib0060"><label>12</label>
<element-citation publication-type="journal" id="sbref0060"><person-group person-group-type="author"><name><surname>Mailles</surname>
<given-names>A.</given-names>
</name>
<name><surname>Blanckaert</surname>
<given-names>K.</given-names>
</name>
<name><surname>Chaud</surname>
<given-names>P.</given-names>
</name>
<name><surname>van der Werf</surname>
<given-names>S.</given-names>
</name>
<name><surname>Lina</surname>
<given-names>B.</given-names>
</name>
<name><surname>Caro</surname>
<given-names>V.</given-names>
</name>
</person-group>
<article-title>First cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infections in France, investigations and implications for the prevention of human-to-human transmission, France, May 2013</article-title>
<source>Euro Surveill</source>
<volume>18</volume>
<year>2013</year>
<comment>pii:20502</comment>
</element-citation>
</ref>
<ref id="bib0065"><label>13</label>
<element-citation publication-type="journal" id="sbref0065"><person-group person-group-type="author"><name><surname>Puzelli</surname>
<given-names>S.</given-names>
</name>
<name><surname>Azzi</surname>
<given-names>A.</given-names>
</name>
<name><surname>Santini</surname>
<given-names>M.G.</given-names>
</name>
<name><surname>Di</surname>
<given-names>M.A.</given-names>
</name>
<name><surname>Facchini</surname>
<given-names>M.</given-names>
</name>
<name><surname>Castrucci</surname>
<given-names>M.R.</given-names>
</name>
</person-group>
<article-title>Investigation of an imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in Florence, Italy, May to June 2013</article-title>
<source>Euro Surveill</source>
<volume>18</volume>
<year>2013</year>
<comment>pii:20564</comment>
</element-citation>
</ref>
<ref id="bib0070"><label>14</label>
<element-citation publication-type="journal" id="sbref0070"><person-group person-group-type="author"><name><surname>Li</surname>
<given-names>F.</given-names>
</name>
</person-group>
<article-title>Evidence for a common evolutionary origin of coronavirus spike protein receptor-binding subunits</article-title>
<source>J Virol</source>
<volume>86</volume>
<year>2012</year>
<fpage>2856</fpage>
<lpage>2858</lpage>
<pub-id pub-id-type="pmid">22205743</pub-id>
</element-citation>
</ref>
<ref id="bib0075"><label>15</label>
<element-citation publication-type="journal" id="sbref0075"><person-group person-group-type="author"><name><surname>Raj</surname>
<given-names>V.S.</given-names>
</name>
<name><surname>Mou</surname>
<given-names>H.</given-names>
</name>
<name><surname>Smits</surname>
<given-names>S.L.</given-names>
</name>
<name><surname>Dekkers</surname>
<given-names>D.H.</given-names>
</name>
<name><surname>Muller</surname>
<given-names>M.A.</given-names>
</name>
<name><surname>Dijkman</surname>
<given-names>R.</given-names>
</name>
</person-group>
<article-title>Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC</article-title>
<source>Nature</source>
<volume>495</volume>
<year>2013</year>
<fpage>251</fpage>
<lpage>254</lpage>
<pub-id pub-id-type="pmid">23486063</pub-id>
</element-citation>
</ref>
<ref id="bib0080"><label>16</label>
<element-citation publication-type="journal" id="sbref0080"><person-group person-group-type="author"><name><surname>Lu</surname>
<given-names>G.</given-names>
</name>
<name><surname>Hu</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
<name><surname>Qi</surname>
<given-names>J.</given-names>
</name>
<name><surname>Gao</surname>
<given-names>F.</given-names>
</name>
<name><surname>Li</surname>
<given-names>Y.</given-names>
</name>
</person-group>
<article-title>Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26</article-title>
<source>Nature</source>
<volume>500</volume>
<year>2013</year>
<fpage>227</fpage>
<lpage>231</lpage>
<pub-id pub-id-type="pmid">23831647</pub-id>
</element-citation>
</ref>
<ref id="bib0085"><label>17</label>
<element-citation publication-type="journal" id="sbref0085"><person-group person-group-type="author"><name><surname>Mou</surname>
<given-names>H.</given-names>
</name>
<name><surname>Raj</surname>
<given-names>V.S.</given-names>
</name>
<name><surname>van Kuppeveld</surname>
<given-names>F.J.</given-names>
</name>
<name><surname>Rottier</surname>
<given-names>P.J.</given-names>
</name>
<name><surname>Haagmans</surname>
<given-names>B.L.</given-names>
</name>
<name><surname>Bosch</surname>
<given-names>B.J.</given-names>
</name>
</person-group>
<article-title>The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies</article-title>
<source>J Virol</source>
<volume>87</volume>
<year>2013</year>
<fpage>9379</fpage>
<lpage>9383</lpage>
<pub-id pub-id-type="pmid">23785207</pub-id>
</element-citation>
</ref>
<ref id="bib0090"><label>18</label>
<element-citation publication-type="journal" id="sbref0090"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Kou</surname>
<given-names>Z.</given-names>
</name>
<name><surname>Ma</surname>
<given-names>C.</given-names>
</name>
<name><surname>Tao</surname>
<given-names>X.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
</person-group>
<article-title>A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines</article-title>
<source>PLoS One</source>
<volume>8</volume>
<year>2013</year>
<fpage>e81587</fpage>
<pub-id pub-id-type="pmid">24324708</pub-id>
</element-citation>
</ref>
<ref id="bib0095"><label>19</label>
<element-citation publication-type="journal" id="sbref0095"><person-group person-group-type="author"><name><surname>Chen</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Rajashankar</surname>
<given-names>K.R.</given-names>
</name>
<name><surname>Yang</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Agnihothram</surname>
<given-names>S.S.</given-names>
</name>
<name><surname>Liu</surname>
<given-names>C.</given-names>
</name>
<name><surname>Lin</surname>
<given-names>Y.L.</given-names>
</name>
</person-group>
<article-title>Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus</article-title>
<source>J Virol</source>
<volume>87</volume>
<year>2013</year>
<fpage>10777</fpage>
<lpage>10783</lpage>
<pub-id pub-id-type="pmid">23903833</pub-id>
</element-citation>
</ref>
<ref id="bib0100"><label>20</label>
<element-citation publication-type="journal" id="sbref0100"><person-group person-group-type="author"><name><surname>Wang</surname>
<given-names>N.</given-names>
</name>
<name><surname>Shi</surname>
<given-names>X.</given-names>
</name>
<name><surname>Jiang</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>S.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>D.</given-names>
</name>
<name><surname>Tong</surname>
<given-names>P.</given-names>
</name>
</person-group>
<article-title>Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4</article-title>
<source>Cell Res</source>
<volume>23</volume>
<year>2013</year>
<fpage>986</fpage>
<lpage>993</lpage>
<pub-id pub-id-type="pmid">23835475</pub-id>
</element-citation>
</ref>
<ref id="bib0105"><label>21</label>
<element-citation publication-type="journal" id="sbref0105"><person-group person-group-type="author"><name><surname>Zhong</surname>
<given-names>N.S.</given-names>
</name>
<name><surname>Zheng</surname>
<given-names>B.J.</given-names>
</name>
<name><surname>Li</surname>
<given-names>Y.M.</given-names>
</name>
<name><surname>Poon</surname>
</name>
<name><surname>Xie</surname>
<given-names>Z.H.</given-names>
</name>
<name><surname>Chan</surname>
<given-names>K.H.</given-names>
</name>
</person-group>
<article-title>Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003</article-title>
<source>Lancet</source>
<volume>362</volume>
<year>2003</year>
<fpage>1353</fpage>
<lpage>1358</lpage>
<pub-id pub-id-type="pmid">14585636</pub-id>
</element-citation>
</ref>
<ref id="bib0110"><label>22</label>
<element-citation publication-type="journal" id="sbref0110"><person-group person-group-type="author"><name><surname>Skowronski</surname>
<given-names>D.M.</given-names>
</name>
<name><surname>Astell</surname>
<given-names>C.</given-names>
</name>
<name><surname>Brunham</surname>
<given-names>R.C.</given-names>
</name>
<name><surname>Low</surname>
<given-names>D.E.</given-names>
</name>
<name><surname>Petric</surname>
<given-names>M.</given-names>
</name>
<name><surname>Roper</surname>
<given-names>R.L.</given-names>
</name>
</person-group>
<article-title>Severe acute respiratory syndrome (SARS): a year in review</article-title>
<source>Annu Rev Med</source>
<volume>56</volume>
<year>2005</year>
<fpage>357</fpage>
<lpage>381</lpage>
<pub-id pub-id-type="pmid">15660517</pub-id>
</element-citation>
</ref>
<ref id="bib0115"><label>23</label>
<element-citation publication-type="journal" id="sbref0115"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>He</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Liu</surname>
<given-names>S.</given-names>
</name>
<name><surname>Zheng</surname>
<given-names>B.J.</given-names>
</name>
<name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
</person-group>
<article-title>The spike protein of SARS-CoV—a target for vaccine and therapeutic development</article-title>
<source>Nat Rev Microbiol</source>
<volume>7</volume>
<year>2009</year>
<fpage>226</fpage>
<lpage>236</lpage>
<pub-id pub-id-type="pmid">19198616</pub-id>
</element-citation>
</ref>
<ref id="bib0120"><label>24</label>
<element-citation publication-type="journal" id="sbref0120"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Chan</surname>
<given-names>C.C.</given-names>
</name>
<name><surname>Sun</surname>
<given-names>S.</given-names>
</name>
<name><surname>Chen</surname>
<given-names>M.</given-names>
</name>
<name><surname>Liu</surname>
<given-names>Z.</given-names>
</name>
</person-group>
<article-title>Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and <italic>E. coli</italic>
cells elicits potent neutralizing antibody and protective immunity</article-title>
<source>Virology</source>
<volume>393</volume>
<year>2009</year>
<fpage>144</fpage>
<lpage>150</lpage>
<pub-id pub-id-type="pmid">19683779</pub-id>
</element-citation>
</ref>
<ref id="bib0125"><label>25</label>
<element-citation publication-type="journal" id="sbref0125"><person-group person-group-type="author"><name><surname>He</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Liu</surname>
<given-names>S.</given-names>
</name>
<name><surname>Kou</surname>
<given-names>Z.</given-names>
</name>
<name><surname>Li</surname>
<given-names>W.</given-names>
</name>
<name><surname>Farzan</surname>
<given-names>M.</given-names>
</name>
</person-group>
<article-title>Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine</article-title>
<source>Biochem Biophys Res Commun</source>
<volume>324</volume>
<year>2004</year>
<fpage>773</fpage>
<lpage>781</lpage>
<pub-id pub-id-type="pmid">15474494</pub-id>
</element-citation>
</ref>
<ref id="bib0130"><label>26</label>
<element-citation publication-type="journal" id="sbref0130"><person-group person-group-type="author"><name><surname>He</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Lu</surname>
<given-names>H.</given-names>
</name>
<name><surname>Siddiqui</surname>
<given-names>P.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
</person-group>
<article-title>Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies</article-title>
<source>J Immunol</source>
<volume>174</volume>
<year>2005</year>
<fpage>4908</fpage>
<lpage>4915</lpage>
<pub-id pub-id-type="pmid">15814718</pub-id>
</element-citation>
</ref>
<ref id="bib0135"><label>27</label>
<element-citation publication-type="journal" id="sbref0135"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Chan</surname>
<given-names>C.C.</given-names>
</name>
<name><surname>Li</surname>
<given-names>L.</given-names>
</name>
<name><surname>He</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
</person-group>
<article-title>A 219-mer CHO-expressing receptor-binding domain of SARS-CoV S protein induces potent immune responses and protective immunity</article-title>
<source>Viral Immunol</source>
<volume>23</volume>
<year>2010</year>
<fpage>211</fpage>
<lpage>219</lpage>
<pub-id pub-id-type="pmid">20374001</pub-id>
</element-citation>
</ref>
<ref id="bib0140"><label>28</label>
<element-citation publication-type="journal" id="sbref0140"><person-group person-group-type="author"><name><surname>Li</surname>
<given-names>F.</given-names>
</name>
<name><surname>Li</surname>
<given-names>W.</given-names>
</name>
<name><surname>Farzan</surname>
<given-names>M.</given-names>
</name>
<name><surname>Harrison</surname>
<given-names>S.C.</given-names>
</name>
</person-group>
<article-title>Structure of SARS coronavirus spike receptor-binding domain complexed with receptor</article-title>
<source>Science</source>
<volume>309</volume>
<year>2005</year>
<fpage>1864</fpage>
<lpage>1868</lpage>
<pub-id pub-id-type="pmid">16166518</pub-id>
</element-citation>
</ref>
<ref id="bib0145"><label>29</label>
<element-citation publication-type="journal" id="sbref0145"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Kou</surname>
<given-names>Z.</given-names>
</name>
<name><surname>Ma</surname>
<given-names>C.</given-names>
</name>
<name><surname>Sun</surname>
<given-names>S.</given-names>
</name>
<name><surname>Poon</surname>
<given-names>V.K.</given-names>
</name>
</person-group>
<article-title>Identification of a receptor-binding domain in the S protein of the novel human coronavirus Middle East respiratory syndrome coronavirus as an essential target for vaccine development</article-title>
<source>J Virol</source>
<volume>87</volume>
<year>2013</year>
<fpage>9939</fpage>
<lpage>9942</lpage>
<pub-id pub-id-type="pmid">23824801</pub-id>
</element-citation>
</ref>
<ref id="bib0150"><label>30</label>
<element-citation publication-type="journal" id="sbref0150"><person-group person-group-type="author"><name><surname>Ma</surname>
<given-names>C.</given-names>
</name>
<name><surname>Li</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Tao</surname>
<given-names>X.</given-names>
</name>
<name><surname>Tseng</surname>
<given-names>C.T.</given-names>
</name>
</person-group>
<article-title>Intranasal vaccination with recombinant receptor-binding domain of MERS-CoV spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal MERS vaccines</article-title>
<source>Vaccine</source>
<volume>32</volume>
<year>2014</year>
<fpage>2100</fpage>
<lpage>2108</lpage>
<pub-id pub-id-type="pmid">24560617</pub-id>
</element-citation>
</ref>
<ref id="bib0155"><label>31</label>
<element-citation publication-type="journal" id="sbref0155"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Yang</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Qiu</surname>
<given-names>H.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>L.</given-names>
</name>
<name><surname>Kou</surname>
<given-names>Z.</given-names>
</name>
</person-group>
<article-title>A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein</article-title>
<source>J Virol</source>
<volume>88</volume>
<year>2014</year>
<fpage>7045</fpage>
<lpage>7053</lpage>
<pub-id pub-id-type="pmid">24719424</pub-id>
</element-citation>
</ref>
<ref id="bib0160"><label>32</label>
<element-citation publication-type="journal" id="sbref0160"><person-group person-group-type="author"><name><surname>Tao</surname>
<given-names>X.</given-names>
</name>
<name><surname>Hill</surname>
<given-names>T.E.</given-names>
</name>
<name><surname>Morimoto</surname>
<given-names>C.</given-names>
</name>
<name><surname>Peters</surname>
<given-names>C.J.</given-names>
</name>
<name><surname>Ksiazek</surname>
<given-names>T.G.</given-names>
</name>
<name><surname>Tseng</surname>
<given-names>C.T.</given-names>
</name>
</person-group>
<article-title>Bilateral entry and release of Middle East respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells</article-title>
<source>J Virol</source>
<volume>87</volume>
<year>2013</year>
<fpage>9953</fpage>
<lpage>9958</lpage>
<pub-id pub-id-type="pmid">23824802</pub-id>
</element-citation>
</ref>
<ref id="bib0165"><label>33</label>
<element-citation publication-type="journal" id="sbref0165"><person-group person-group-type="author"><name><surname>Zhang</surname>
<given-names>N.</given-names>
</name>
<name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<name><surname>Du</surname>
<given-names>L.</given-names>
</name>
</person-group>
<article-title>Current advancements and potential strategies in the development of MERS-CoV vaccines</article-title>
<source>Expert Rev Vaccines</source>
<volume>13</volume>
<year>2014</year>
<fpage>761</fpage>
<lpage>774</lpage>
<pub-id pub-id-type="pmid">24766432</pub-id>
</element-citation>
</ref>
<ref id="bib0170"><label>34</label>
<element-citation publication-type="journal" id="sbref0170"><person-group person-group-type="author"><name><surname>Song</surname>
<given-names>F.</given-names>
</name>
<name><surname>Fux</surname>
<given-names>R.</given-names>
</name>
<name><surname>Provacia</surname>
<given-names>L.B.</given-names>
</name>
<name><surname>Volz</surname>
<given-names>A.</given-names>
</name>
<name><surname>Eickmann</surname>
<given-names>M.</given-names>
</name>
<name><surname>Becker</surname>
<given-names>S.</given-names>
</name>
</person-group>
<article-title>Middle East respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies</article-title>
<source>J Virol</source>
<volume>87</volume>
<year>2013</year>
<fpage>11950</fpage>
<lpage>11954</lpage>
<pub-id pub-id-type="pmid">23986586</pub-id>
</element-citation>
</ref>
<ref id="bib0175"><label>35</label>
<element-citation publication-type="journal" id="sbref0175"><person-group person-group-type="author"><name><surname>Agnihothram</surname>
<given-names>S.</given-names>
</name>
<name><surname>Gopal</surname>
<given-names>R.</given-names>
</name>
<name><surname>Yount</surname>
<given-names>B.L.</given-names>
<suffix>Jr.</suffix>
</name>
<name><surname>Donaldson</surname>
<given-names>E.F.</given-names>
</name>
<name><surname>Menachery</surname>
<given-names>V.D.</given-names>
</name>
<name><surname>Graham</surname>
<given-names>R.L.</given-names>
</name>
</person-group>
<article-title>Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses</article-title>
<source>J Infect Dis</source>
<volume>209</volume>
<year>2014</year>
<fpage>995</fpage>
<lpage>1006</lpage>
<pub-id pub-id-type="pmid">24253287</pub-id>
</element-citation>
</ref>
<ref id="bib0180"><label>36</label>
<element-citation publication-type="journal" id="sbref0180"><person-group person-group-type="author"><name><surname>Coleman</surname>
<given-names>C.M.</given-names>
</name>
<name><surname>Liu</surname>
<given-names>Y.V.</given-names>
</name>
<name><surname>Mu</surname>
<given-names>H.</given-names>
</name>
<name><surname>Taylor</surname>
<given-names>J.K.</given-names>
</name>
<name><surname>Massare</surname>
<given-names>M.</given-names>
</name>
<name><surname>Flyer</surname>
<given-names>D.C.</given-names>
</name>
</person-group>
<article-title>Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice</article-title>
<source>Vaccine</source>
<volume>32</volume>
<year>2014</year>
<fpage>3169</fpage>
<lpage>3174</lpage>
<pub-id pub-id-type="pmid">24736006</pub-id>
</element-citation>
</ref>
<ref id="bib0185"><label>37</label>
<element-citation publication-type="journal" id="sbref0185"><person-group person-group-type="author"><name><surname>Martyn</surname>
<given-names>J.C.</given-names>
</name>
<name><surname>Cardin</surname>
<given-names>A.J.</given-names>
</name>
<name><surname>Wines</surname>
<given-names>B.D.</given-names>
</name>
<name><surname>Cendron</surname>
<given-names>A.</given-names>
</name>
<name><surname>Li</surname>
<given-names>S.</given-names>
</name>
<name><surname>Mackenzie</surname>
<given-names>J.</given-names>
</name>
</person-group>
<article-title>Surface display of IgG Fc on baculovirus vectors enhances binding to antigen-presenting cells and cell lines expressing Fc receptors</article-title>
<source>Arch Virol</source>
<volume>154</volume>
<year>2009</year>
<fpage>1129</fpage>
<lpage>1138</lpage>
<pub-id pub-id-type="pmid">19557497</pub-id>
</element-citation>
</ref>
<ref id="bib0190"><label>38</label>
<element-citation publication-type="journal" id="sbref0190"><person-group person-group-type="author"><name><surname>Chen</surname>
<given-names>H.</given-names>
</name>
<name><surname>Xu</surname>
<given-names>X.</given-names>
</name>
<name><surname>Jones</surname>
<given-names>I.M.</given-names>
</name>
</person-group>
<article-title>Immunogenicity of the outer domain of a HIV-1 clade C gp120</article-title>
<source>Retrovirology</source>
<volume>4</volume>
<year>2007</year>
<fpage>33</fpage>
<pub-id pub-id-type="pmid">17509143</pub-id>
</element-citation>
</ref>
<ref id="bib0195"><label>39</label>
<element-citation publication-type="journal" id="sbref0195"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Sun</surname>
<given-names>S.</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>X.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>X.</given-names>
</name>
<name><surname>Guo</surname>
<given-names>Y.</given-names>
</name>
</person-group>
<article-title>A critical HA1 neutralizing domain of H5N1influenza in an optimal conformation induces strong cross-protection</article-title>
<source>PLoS One</source>
<volume>8</volume>
<year>2013</year>
<fpage>e53568</fpage>
<pub-id pub-id-type="pmid">23320093</pub-id>
</element-citation>
</ref>
<ref id="bib0200"><label>40</label>
<element-citation publication-type="journal" id="sbref0200"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Leung</surname>
<given-names>V.H.</given-names>
</name>
<name><surname>Zhang</surname>
<given-names>X.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>J.</given-names>
</name>
<name><surname>Chen</surname>
<given-names>M.</given-names>
</name>
<name><surname>He</surname>
<given-names>W.</given-names>
</name>
</person-group>
<article-title>A recombinant vaccine of H5N1 HA1 fused with foldon and human IgG Fc induced complete cross-clade protection against divergent H5N1 viruses</article-title>
<source>PLoS One</source>
<volume>6</volume>
<year>2011</year>
<fpage>e16555</fpage>
<pub-id pub-id-type="pmid">21304591</pub-id>
</element-citation>
</ref>
<ref id="bib0205"><label>41</label>
<element-citation publication-type="journal" id="sbref0205"><person-group person-group-type="author"><name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>He</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Guo</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Zheng</surname>
<given-names>B.J.</given-names>
</name>
<name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
</person-group>
<article-title>Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model</article-title>
<source>Vaccine</source>
<volume>25</volume>
<year>2007</year>
<fpage>2832</fpage>
<lpage>2838</lpage>
<pub-id pub-id-type="pmid">17092615</pub-id>
</element-citation>
</ref>
<ref id="bib0210"><label>42</label>
<element-citation publication-type="journal" id="sbref0210"><person-group person-group-type="author"><name><surname>Li</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Qiu</surname>
<given-names>H.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>L.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
</person-group>
<article-title>A recombinant protein containing highly conserved hemagglutinin residues 81-122 of influenza H5N1 induces strong humoral and mucosal immune responses</article-title>
<source>BioSci Trends</source>
<volume>7</volume>
<year>2013</year>
<fpage>129</fpage>
<lpage>137</lpage>
<pub-id pub-id-type="pmid">23836036</pub-id>
</element-citation>
</ref>
<ref id="bib0215"><label>43</label>
<element-citation publication-type="journal" id="sbref0215"><person-group person-group-type="author"><name><surname>Jiang</surname>
<given-names>L.</given-names>
</name>
<name><surname>Wang</surname>
<given-names>N.</given-names>
</name>
<name><surname>Zuo</surname>
<given-names>T.</given-names>
</name>
<name><surname>Shi</surname>
<given-names>X.</given-names>
</name>
<name><surname>Poon</surname>
<given-names>K.M.</given-names>
</name>
<name><surname>Wu</surname>
<given-names>Y.</given-names>
</name>
</person-group>
<article-title>Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein</article-title>
<source>Sci Transl Med</source>
<volume>6</volume>
<year>2014</year>
<fpage>234ra59</fpage>
</element-citation>
</ref>
<ref id="bib0220"><label>44</label>
<element-citation publication-type="journal" id="sbref0220"><person-group person-group-type="author"><name><surname>Tang</surname>
<given-names>X.C.</given-names>
</name>
<name><surname>Agnihothram</surname>
<given-names>S.S.</given-names>
</name>
<name><surname>Jiao</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Stanhope</surname>
<given-names>J.</given-names>
</name>
<name><surname>Graham</surname>
<given-names>R.L.</given-names>
</name>
<name><surname>Peterson</surname>
<given-names>E.C.</given-names>
</name>
</person-group>
<article-title>Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution</article-title>
<source>Proc Natl Acad Sci USA</source>
<volume>111</volume>
<year>2014</year>
<fpage>E2018</fpage>
<lpage>E2026</lpage>
<pub-id pub-id-type="pmid">24778221</pub-id>
</element-citation>
</ref>
<ref id="bib0225"><label>45</label>
<element-citation publication-type="journal" id="sbref0225"><person-group person-group-type="author"><name><surname>Ying</surname>
<given-names>T.</given-names>
</name>
<name><surname>Du</surname>
<given-names>L.</given-names>
</name>
<name><surname>Ju</surname>
<given-names>T.W.</given-names>
</name>
<name><surname>Prabakaran</surname>
<given-names>P.</given-names>
</name>
<name><surname>Lau</surname>
<given-names>C.C.</given-names>
</name>
<name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
</person-group>
<article-title>Exceptionally potent neutralization of middle East respiratory syndrome coronavirus by human monoclonal antibodies</article-title>
<source>J Virol</source>
<volume>88</volume>
<year>2014</year>
<fpage>7796</fpage>
<lpage>7805</lpage>
<pub-id pub-id-type="pmid">24789777</pub-id>
</element-citation>
</ref>
<ref id="bib0230"><label>46</label>
<element-citation publication-type="journal" id="sbref0230"><person-group person-group-type="author"><name><surname>Czub</surname>
<given-names>M.</given-names>
</name>
<name><surname>Weingartl</surname>
<given-names>H.</given-names>
</name>
<name><surname>Czub</surname>
<given-names>S.</given-names>
</name>
<name><surname>He</surname>
<given-names>R.</given-names>
</name>
<name><surname>Cao</surname>
<given-names>J.</given-names>
</name>
</person-group>
<article-title>Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets</article-title>
<source>Vaccine</source>
<volume>23</volume>
<year>2005</year>
<fpage>2273</fpage>
<lpage>2279</lpage>
<pub-id pub-id-type="pmid">15755610</pub-id>
</element-citation>
</ref>
<ref id="bib0235"><label>47</label>
<element-citation publication-type="journal" id="sbref0235"><person-group person-group-type="author"><name><surname>He</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Wu</surname>
<given-names>H.</given-names>
</name>
<name><surname>Luo</surname>
<given-names>B.</given-names>
</name>
<name><surname>Chen</surname>
<given-names>J.</given-names>
</name>
<name><surname>Li</surname>
<given-names>W.</given-names>
</name>
</person-group>
<article-title>Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines</article-title>
<source>J Immunol</source>
<volume>173</volume>
<year>2004</year>
<fpage>4050</fpage>
<lpage>4057</lpage>
<pub-id pub-id-type="pmid">15356154</pub-id>
</element-citation>
</ref>
<ref id="bib0240"><label>48</label>
<element-citation publication-type="journal" id="sbref0240"><person-group person-group-type="author"><name><surname>Weingartl</surname>
<given-names>H.</given-names>
</name>
<name><surname>Czub</surname>
<given-names>M.</given-names>
</name>
<name><surname>Czub</surname>
<given-names>S.</given-names>
</name>
<name><surname>Neufeld</surname>
<given-names>J.</given-names>
</name>
<name><surname>Marszal</surname>
<given-names>P.</given-names>
</name>
<name><surname>Gren</surname>
<given-names>J.</given-names>
</name>
</person-group>
<article-title>Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets</article-title>
<source>J Virol</source>
<volume>78</volume>
<year>2004</year>
<fpage>12672</fpage>
<lpage>12676</lpage>
<pub-id pub-id-type="pmid">15507655</pub-id>
</element-citation>
</ref>
<ref id="bib0245"><label>49</label>
<element-citation publication-type="journal" id="sbref0245"><person-group person-group-type="author"><name><surname>de</surname>
<given-names>W.E.</given-names>
</name>
<name><surname>Rasmussen</surname>
<given-names>A.L.</given-names>
</name>
<name><surname>Falzarano</surname>
<given-names>D.</given-names>
</name>
<name><surname>Bushmaker</surname>
<given-names>T.</given-names>
</name>
<name><surname>Feldmann</surname>
<given-names>F.</given-names>
</name>
<name><surname>Brining</surname>
<given-names>D.L.</given-names>
</name>
</person-group>
<article-title>Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques</article-title>
<source>Proc Natl Acad Sci USA</source>
<volume>110</volume>
<year>2013</year>
<fpage>16598</fpage>
<lpage>16603</lpage>
<pub-id pub-id-type="pmid">24062443</pub-id>
</element-citation>
</ref>
<ref id="bib0250"><label>50</label>
<element-citation publication-type="journal" id="sbref0250"><person-group person-group-type="author"><name><surname>Yao</surname>
<given-names>Y.</given-names>
</name>
<name><surname>Bao</surname>
<given-names>L.</given-names>
</name>
<name><surname>Deng</surname>
<given-names>W.</given-names>
</name>
<name><surname>Xu</surname>
<given-names>L.</given-names>
</name>
<name><surname>Li</surname>
<given-names>F.</given-names>
</name>
<name><surname>Lv</surname>
<given-names>Q.</given-names>
</name>
</person-group>
<article-title>An animal model of MERS produced by infection of rhesus macaques with MERS coronavirus</article-title>
<source>J Infect Dis</source>
<volume>209</volume>
<year>2014</year>
<fpage>236</fpage>
<lpage>242</lpage>
<pub-id pub-id-type="pmid">24218506</pub-id>
</element-citation>
</ref>
<ref id="bib0255"><label>51</label>
<element-citation publication-type="journal" id="sbref0255"><person-group person-group-type="author"><name><surname>de</surname>
<given-names>W.E.</given-names>
</name>
<name><surname>Prescott</surname>
<given-names>J.</given-names>
</name>
<name><surname>Baseler</surname>
<given-names>L.</given-names>
</name>
<name><surname>Bushmaker</surname>
<given-names>T.</given-names>
</name>
<name><surname>Thomas</surname>
<given-names>T.</given-names>
</name>
<name><surname>Lackemeyer</surname>
<given-names>M.G.</given-names>
</name>
</person-group>
<article-title>The Middle East respiratory syndrome coronavirus (MERS-CoV) does not replicate in Syrian hamsters</article-title>
<source>PLoS One</source>
<volume>8</volume>
<year>2013</year>
<fpage>e69127</fpage>
<pub-id pub-id-type="pmid">23844250</pub-id>
</element-citation>
</ref>
<ref id="bib0260"><label>52</label>
<element-citation publication-type="journal" id="sbref0260"><person-group person-group-type="author"><name><surname>Coleman</surname>
<given-names>C.M.</given-names>
</name>
<name><surname>Matthews</surname>
<given-names>K.L.</given-names>
</name>
<name><surname>Goicochea</surname>
<given-names>L.</given-names>
</name>
<name><surname>Frieman</surname>
<given-names>M.B.</given-names>
</name>
</person-group>
<article-title>Wild type and innate immune deficient mice are not susceptible to the Middle East respiratory syndrome coronavirus</article-title>
<source>J Gen Virol</source>
<volume>95</volume>
<year>2014</year>
<fpage>408</fpage>
<lpage>412</lpage>
<pub-id pub-id-type="pmid">24197535</pub-id>
</element-citation>
</ref>
<ref id="bib0265"><label>53</label>
<element-citation publication-type="journal" id="sbref0265"><person-group person-group-type="author"><name><surname>Martina</surname>
<given-names>B.E.</given-names>
</name>
<name><surname>Haagmans</surname>
<given-names>B.L.</given-names>
</name>
<name><surname>Kuiken</surname>
<given-names>T.</given-names>
</name>
<name><surname>Fouchier</surname>
<given-names>R.A.</given-names>
</name>
<name><surname>Rimmelzwaan</surname>
<given-names>G.F.</given-names>
</name>
<name><surname>Van</surname>
<given-names>A.G.</given-names>
</name>
</person-group>
<article-title>Virology: SARS virus infection of cats and ferrets</article-title>
<source>Nature</source>
<volume>425</volume>
<year>2003</year>
<fpage>915</fpage>
<pub-id pub-id-type="pmid">14586458</pub-id>
</element-citation>
</ref>
<ref id="bib0270"><label>54</label>
<element-citation publication-type="journal" id="sbref0270"><person-group person-group-type="author"><name><surname>Roberts</surname>
<given-names>A.</given-names>
</name>
<name><surname>Vogel</surname>
<given-names>L.</given-names>
</name>
<name><surname>Guarner</surname>
<given-names>J.</given-names>
</name>
<name><surname>Hayes</surname>
<given-names>N.</given-names>
</name>
<name><surname>Murphy</surname>
<given-names>B.</given-names>
</name>
<name><surname>Zaki</surname>
<given-names>S.</given-names>
</name>
</person-group>
<article-title>Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters</article-title>
<source>J Virol</source>
<volume>79</volume>
<year>2005</year>
<fpage>503</fpage>
<lpage>511</lpage>
<pub-id pub-id-type="pmid">15596843</pub-id>
</element-citation>
</ref>
<ref id="bib0275"><label>55</label>
<element-citation publication-type="journal" id="sbref0275"><person-group person-group-type="author"><name><surname>Zhao</surname>
<given-names>J.</given-names>
</name>
<name><surname>Li</surname>
<given-names>K.</given-names>
</name>
<name><surname>Wohlford-Lenane</surname>
<given-names>C.</given-names>
</name>
<name><surname>Agnihothram</surname>
<given-names>S.S.</given-names>
</name>
<name><surname>Fett</surname>
<given-names>C.</given-names>
</name>
<name><surname>Zhao</surname>
<given-names>J.</given-names>
</name>
</person-group>
<article-title>Rapid generation of a mouse model for Middle East respiratory syndrome</article-title>
<source>Proc Natl Acad Sci USA</source>
<volume>111</volume>
<year>2014</year>
<fpage>4970</fpage>
<lpage>4975</lpage>
<pub-id pub-id-type="pmid">24599590</pub-id>
</element-citation>
</ref>
</ref-list>
<ack id="ack0005"><title>Acknowledgements</title>
<p>This study was supported by <funding-source id="gs0005">NIH</funding-source>
grant R21AI109094, the intramural fund of <funding-source id="gs2">New York Blood Center</funding-source>
NYB000068 (to L. Du and S. Jiang), and <funding-source id="gs0010">NIH</funding-source>
grant R01AI089728 (to F. Li).</p>
</ack>
</back>
</pmc>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001165 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 001165 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= MersV1 |flux= Pmc |étape= Corpus |type= RBID |clé= |texte= }}
![]() | This area was generated with Dilib version V0.6.33. | ![]() |