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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Immunodominant SARS Coronavirus Epitopes in Humans
Elicited both Enhancing and Neutralizing Effects on Infection in Non-human
Primates</title><author><name sortKey="Wang, Qidi" sort="Wang, Qidi" uniqKey="Wang Q" first="Qidi" last="Wang">Qidi Wang</name><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Lianfeng" sort="Zhang, Lianfeng" uniqKey="Zhang L" first="Lianfeng" last="Zhang">Lianfeng Zhang</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Kuwahara, Kazuhiko" sort="Kuwahara, Kazuhiko" uniqKey="Kuwahara K" first="Kazuhiko" last="Kuwahara">Kazuhiko Kuwahara</name><affiliation><nlm:aff id="aff4">Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></nlm:aff></affiliation></author><author><name sortKey="Li, Li" sort="Li, Li" uniqKey="Li L" first="Li" last="Li">Li Li</name><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Zijie" sort="Liu, Zijie" uniqKey="Liu Z" first="Zijie" last="Liu">Zijie Liu</name><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Li, Taisheng" sort="Li, Taisheng" uniqKey="Li T" first="Taisheng" last="Li">Taisheng Li</name><affiliation><nlm:aff id="aff5">Department of Infectious Disease, Peking Union Medical College Hospital and AIDS Research Center,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100071,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Zhu, Hua" sort="Zhu, Hua" uniqKey="Zhu H" first="Hua" last="Zhu">Hua Zhu</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Jiangning" sort="Liu, Jiangning" uniqKey="Liu J" first="Jiangning" last="Liu">Jiangning Liu</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Xu, Yanfeng" sort="Xu, Yanfeng" uniqKey="Xu Y" first="Yanfeng" last="Xu">Yanfeng Xu</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Xie, Jing" sort="Xie, Jing" uniqKey="Xie J" first="Jing" last="Xie">Jing Xie</name><affiliation><nlm:aff id="aff5">Department of Infectious Disease, Peking Union Medical College Hospital and AIDS Research Center,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100071,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Morioka, Hiroshi" sort="Morioka, Hiroshi" uniqKey="Morioka H" first="Hiroshi" last="Morioka">Hiroshi Morioka</name><affiliation><nlm:aff id="aff4">Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></nlm:aff></affiliation></author><author><name sortKey="Sakaguchi, Nobuo" sort="Sakaguchi, Nobuo" uniqKey="Sakaguchi N" first="Nobuo" last="Sakaguchi">Nobuo Sakaguchi</name><affiliation><nlm:aff id="aff4">Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff6">WPI Immunology Frontier Research Center,<institution>Osaka University</institution>, 3-1 Yamada-oka, Suita, Osaka 565-0871,<country>Japan</country></nlm:aff></affiliation></author><author><name sortKey="Qin, Chuan" sort="Qin, Chuan" uniqKey="Qin C" first="Chuan" last="Qin">Chuan Qin</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Gang" sort="Liu, Gang" uniqKey="Liu G" first="Gang" last="Liu">Gang Liu</name><affiliation><nlm:aff id="aff1">Tsinghua-Peking Center for Life Sciences & School of Pharmaceutical Sciences,<institution>Tsinghua University</institution>, Haidian District, Beijing 100084,<country>People’s Republic of China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27627203</idno><idno type="pmc">7075522</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075522</idno><idno type="RBID">PMC:7075522</idno><idno type="doi">10.1021/acsinfecdis.6b00006</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000029</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000029</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Immunodominant SARS Coronavirus Epitopes in Humans
Elicited both Enhancing and Neutralizing Effects on Infection in Non-human
Primates</title><author><name sortKey="Wang, Qidi" sort="Wang, Qidi" uniqKey="Wang Q" first="Qidi" last="Wang">Qidi Wang</name><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Lianfeng" sort="Zhang, Lianfeng" uniqKey="Zhang L" first="Lianfeng" last="Zhang">Lianfeng Zhang</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Kuwahara, Kazuhiko" sort="Kuwahara, Kazuhiko" uniqKey="Kuwahara K" first="Kazuhiko" last="Kuwahara">Kazuhiko Kuwahara</name><affiliation><nlm:aff id="aff4">Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></nlm:aff></affiliation></author><author><name sortKey="Li, Li" sort="Li, Li" uniqKey="Li L" first="Li" last="Li">Li Li</name><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Zijie" sort="Liu, Zijie" uniqKey="Liu Z" first="Zijie" last="Liu">Zijie Liu</name><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Li, Taisheng" sort="Li, Taisheng" uniqKey="Li T" first="Taisheng" last="Li">Taisheng Li</name><affiliation><nlm:aff id="aff5">Department of Infectious Disease, Peking Union Medical College Hospital and AIDS Research Center,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100071,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Zhu, Hua" sort="Zhu, Hua" uniqKey="Zhu H" first="Hua" last="Zhu">Hua Zhu</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Jiangning" sort="Liu, Jiangning" uniqKey="Liu J" first="Jiangning" last="Liu">Jiangning Liu</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Xu, Yanfeng" sort="Xu, Yanfeng" uniqKey="Xu Y" first="Yanfeng" last="Xu">Yanfeng Xu</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Xie, Jing" sort="Xie, Jing" uniqKey="Xie J" first="Jing" last="Xie">Jing Xie</name><affiliation><nlm:aff id="aff5">Department of Infectious Disease, Peking Union Medical College Hospital and AIDS Research Center,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100071,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Morioka, Hiroshi" sort="Morioka, Hiroshi" uniqKey="Morioka H" first="Hiroshi" last="Morioka">Hiroshi Morioka</name><affiliation><nlm:aff id="aff4">Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></nlm:aff></affiliation></author><author><name sortKey="Sakaguchi, Nobuo" sort="Sakaguchi, Nobuo" uniqKey="Sakaguchi N" first="Nobuo" last="Sakaguchi">Nobuo Sakaguchi</name><affiliation><nlm:aff id="aff4">Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff6">WPI Immunology Frontier Research Center,<institution>Osaka University</institution>, 3-1 Yamada-oka, Suita, Osaka 565-0871,<country>Japan</country></nlm:aff></affiliation></author><author><name sortKey="Qin, Chuan" sort="Qin, Chuan" uniqKey="Qin C" first="Chuan" last="Qin">Chuan Qin</name><affiliation><nlm:aff id="aff3">Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Gang" sort="Liu, Gang" uniqKey="Liu G" first="Gang" last="Liu">Gang Liu</name><affiliation><nlm:aff id="aff1">Tsinghua-Peking Center for Life Sciences & School of Pharmaceutical Sciences,<institution>Tsinghua University</institution>, Haidian District, Beijing 100084,<country>People’s Republic of China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></nlm:aff></affiliation></author></analytic><series><title level="j">ACS Infectious Diseases</title><idno type="eISSN">2373-8227</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="id6b00006_0011" id="ab-tgr1"></graphic></p><p>Severe
acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV)
and has the potential to threaten global public health and socioeconomic
stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV
infection in vitro and in non-human primates clouds the prospects
for a safe vaccine. Using antibodies from SARS patients, we identified
and characterized SARS-CoV B-cell peptide epitopes with disparate
functions. In rhesus macaques, the spike glycoprotein peptides S<sub>471–503</sub>, S<sub>604–625</sub>, and S<sub>1164–1191</sub> elicited antibodies that efficiently prevented infection in non-human
primates. In contrast, peptide S<sub>597–603</sub> induced
antibodies that enhanced infection both in vitro and in non-human
primates by using an epitope sequence-dependent (ESD) mechanism. This
peptide exhibited a high level of serological reactivity (64%), which
resulted from the additive responses of two tandem epitopes (S<sub>597–603</sub> and S<sub>604–625</sub>) and a long-term
human B-cell memory response with antisera from convalescent SARS
patients. Thus, peptide-based vaccines against SARS-CoV could be engineered
to avoid ADE via elimination of the S<sub>597–603</sub> epitope.
We provide herein an alternative strategy to prepare a safe and effective
vaccine for ADE of viral infection by identifying and eliminating
epitope sequence-dependent enhancement of viral infection.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Hilgenfeld, R" uniqKey="Hilgenfeld R">R. Hilgenfeld</name></author><author><name sortKey="Peiris, M" uniqKey="Peiris M">M. Peiris</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author><author><name sortKey="Gunther, S" uniqKey="Gunther S">S. Günther</name></author><author><name sortKey="Preiser, W" uniqKey="Preiser W">W. Preiser</name></author><author><name sortKey="Van Der Werf, S" uniqKey="Van Der Werf S">S. van der Werf</name></author><author><name sortKey="Brodt, H R" uniqKey="Brodt H">H. R. Brodt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z. Shi</name></author><author><name sortKey="Yu, M" uniqKey="Yu M">M. Yu</name></author><author><name sortKey="Ren, W" uniqKey="Ren W">W. Ren</name></author><author><name sortKey="Smith, C" uniqKey="Smith C">C. Smith</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y. Guan</name></author><author><name sortKey="Zheng, B J" uniqKey="Zheng B">B. J. Zheng</name></author><author><name sortKey="He, Y Q" uniqKey="He Y">Y. Q. He</name></author><author><name sortKey="Liu, X L" uniqKey="Liu X">X. L. Liu</name></author><author><name sortKey="Zhuang, Z X" uniqKey="Zhuang Z">Z. X. Zhuang</name></author><author><name sortKey="Cheung, C L" uniqKey="Cheung C">C. L. Cheung</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Marra, M A" uniqKey="Marra M">M. A. Marra</name></author><author><name sortKey="Jones, S J" uniqKey="Jones S">S. J. Jones</name></author><author><name sortKey="Astell, C R" uniqKey="Astell C">C. R. Astell</name></author><author><name sortKey="Holt, R A" uniqKey="Holt R">R. A. Holt</name></author><author><name sortKey="Brooks Wilson, A" uniqKey="Brooks Wilson A">A. Brooks-Wilson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rota, P A" uniqKey="Rota P">P. A. Rota</name></author><author><name sortKey="Oberste, M S" uniqKey="Oberste M">M. S. Oberste</name></author><author><name sortKey="Monroe, S S" uniqKey="Monroe S">S. S. Monroe</name></author><author><name sortKey="Nix, W A" uniqKey="Nix W">W. A. Nix</name></author><author><name sortKey="Campagnoli, R" uniqKey="Campagnoli R">R. Campagnoli</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Moore, M J" uniqKey="Moore M">M. J. Moore</name></author><author><name sortKey="Vasilieva, N" uniqKey="Vasilieva N">N. Vasilieva</name></author><author><name sortKey="Sui, J" uniqKey="Sui J">J. Sui</name></author><author><name sortKey="Wong, S K" uniqKey="Wong S">S. K. Wong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jeffers, S A" uniqKey="Jeffers S">S. A. Jeffers</name></author><author><name sortKey="Tusell, S M" uniqKey="Tusell S">S. M. Tusell</name></author><author><name sortKey="Gillim Ross, L" uniqKey="Gillim Ross L">L. Gillim-Ross</name></author><author><name sortKey="Hemmila, E M" uniqKey="Hemmila E">E. M. Hemmila</name></author><author><name sortKey="Achenbach, J E" uniqKey="Achenbach J">J. E. Achenbach</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, B" uniqKey="Wang B">B. Wang</name></author><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Jiang, X" uniqKey="Jiang X">X. Jiang</name></author><author><name sortKey="Zhang, M" uniqKey="Zhang M">M. Zhang</name></author><author><name sortKey="Wan, T" uniqKey="Wan T">T. Wan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Y D" uniqKey="Wang Y">Y. D. Wang</name></author><author><name sortKey="Sin, W Y" uniqKey="Sin W">W. Y. Sin</name></author><author><name sortKey="Xu, G B" uniqKey="Xu G">G. B. Xu</name></author><author><name sortKey="Yang, H H" uniqKey="Yang H">H. H. Yang</name></author><author><name sortKey="Wong, T Y" uniqKey="Wong T">T. Y. Wong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Roberts, A" uniqKey="Roberts A">A. Roberts</name></author><author><name sortKey="Wood, J" uniqKey="Wood J">J. Wood</name></author><author><name sortKey="Subbarao, K" uniqKey="Subbarao K">K. Subbarao</name></author><author><name sortKey="Ferguson, M" uniqKey="Ferguson M">M. Ferguson</name></author><author><name sortKey="Wood, D" uniqKey="Wood D">D. Wood</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gillim Ross, L" uniqKey="Gillim Ross L">L. Gillim-Ross</name></author><author><name sortKey="Subbarao, K" uniqKey="Subbarao K">K. Subbarao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Graham, R L" uniqKey="Graham R">R. L. Graham</name></author><author><name sortKey="Donaldson, E F" uniqKey="Donaldson E">E. F. Donaldson</name></author><author><name sortKey="Baric, R S" uniqKey="Baric R">R. S. Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, Z Y" uniqKey="Yang Z">Z. Y. Yang</name></author><author><name sortKey="Werner, H C" uniqKey="Werner H">H. C. Werner</name></author><author><name sortKey="Kong, W P" uniqKey="Kong W">W. P. Kong</name></author><author><name sortKey="Leung, K" uniqKey="Leung K">K. Leung</name></author><author><name sortKey="Traggiai, E" uniqKey="Traggiai E">E. Traggiai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dandekar, A A" uniqKey="Dandekar A">A. A. Dandekar</name></author><author><name sortKey="Perlman, S" uniqKey="Perlman S">S. Perlman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Subbarao, K" uniqKey="Subbarao K">K. Subbarao</name></author><author><name sortKey="Roberts, A" uniqKey="Roberts A">A. Roberts</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taylor, D R" uniqKey="Taylor D">D. R. Taylor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Takada, A" uniqKey="Takada A">A. Takada</name></author><author><name sortKey="Kawaoka, Y" uniqKey="Kawaoka Y">Y. Kawaoka</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tirado, S M C" uniqKey="Tirado S">S. M. C. Tirado</name></author><author><name sortKey="Yoon, K J" uniqKey="Yoon K">K. J. Yoon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gorlani, A" uniqKey="Gorlani A">A. Gorlani</name></author><author><name sortKey="Forthal, D N" uniqKey="Forthal D">D. N. Forthal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sullivan, N" uniqKey="Sullivan N">N. Sullivan</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y. Sun</name></author><author><name sortKey="Binley, J" uniqKey="Binley J">J. Binley</name></author><author><name sortKey="Lee, J" uniqKey="Lee J">J. Lee</name></author><author><name sortKey="Barbas, C F" uniqKey="Barbas C">C. F. Barbas</name></author><author><name sortKey="Parren, P W" uniqKey="Parren P">P. W. Parren</name></author><author><name sortKey="Burton, D R" uniqKey="Burton D">D. R. Burton</name></author><author><name sortKey="Sodroski, J" uniqKey="Sodroski J">J. Sodroski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guillon, C" uniqKey="Guillon C">C. Guillon</name></author><author><name sortKey="Schutten, M" uniqKey="Schutten M">M. Schutten</name></author><author><name sortKey="Boers, P H" uniqKey="Boers P">P. H. Boers</name></author><author><name sortKey="Gruters, R A" uniqKey="Gruters R">R. A. Gruters</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A. D. Osterhaus</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gorlani, A" uniqKey="Gorlani A">A. Gorlani</name></author><author><name sortKey="Forthal, D N" uniqKey="Forthal D">D. N. Forthal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Halstead, S B" uniqKey="Halstead S">S. B. Halstead</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goncalvez, A P" uniqKey="Goncalvez A">A. P. Goncalvez</name></author><author><name sortKey="Engle, R E" uniqKey="Engle R">R. E. Engle</name></author><author><name sortKey="Claire, M" uniqKey="Claire M">M. Claire</name></author><author><name sortKey="Purcell, R H" uniqKey="Purcell R">R. H. Purcell</name></author><author><name sortKey="Lai, C J" uniqKey="Lai C">C. J. Lai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pierson, T C" uniqKey="Pierson T">T. C. Pierson</name></author><author><name sortKey="Xu, Q" uniqKey="Xu Q">Q. Xu</name></author><author><name sortKey="Nelson, S" uniqKey="Nelson S">S. Nelson</name></author><author><name sortKey="Oliphant, T" uniqKey="Oliphant T">T. Oliphant</name></author><author><name sortKey="Grant, E" uniqKey="Grant E">E. Grant</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taylor, A" uniqKey="Taylor A">A. Taylor</name></author><author><name sortKey="Foo, S S" uniqKey="Foo S">S. S. Foo</name></author><author><name sortKey="Bruzzone, R" uniqKey="Bruzzone R">R. Bruzzone</name></author><author><name sortKey="Dinh, L V" uniqKey="Dinh L">L. V. Dinh</name></author><author><name sortKey="King, N J" uniqKey="King N">N. J. King</name></author><author><name sortKey="Mahalingam, S" uniqKey="Mahalingam S">S. Mahalingam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pierson, T C" uniqKey="Pierson T">T. C. Pierson</name></author><author><name sortKey="Sanchez, M D" uniqKey="Sanchez M">M. D. Sánchez</name></author><author><name sortKey="Puffer, B A" uniqKey="Puffer B">B. A. Puffer</name></author><author><name sortKey="Ahmed, A A" uniqKey="Ahmed A">A. A. Ahmed</name></author><author><name sortKey="Geiss, B J" uniqKey="Geiss B">B. J. Geiss</name></author><author><name sortKey="Valentine, L E" uniqKey="Valentine L">L. E. Valentine</name></author><author><name sortKey="Altamura, L A" uniqKey="Altamura L">L. A. Altamura</name></author><author><name sortKey="Diamond, M S" uniqKey="Diamond M">M. S. Diamond</name></author><author><name sortKey="Doms, R W" uniqKey="Doms R">R. W. Doms</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mehlhop, E" uniqKey="Mehlhop E">E. Mehlhop</name></author><author><name sortKey="Ansarah Sobrinho, C" uniqKey="Ansarah Sobrinho C">C. Ansarah-Sobrinho</name></author><author><name sortKey="Johnson, S" uniqKey="Johnson S">S. Johnson</name></author><author><name sortKey="Engle, M" uniqKey="Engle M">M. Engle</name></author><author><name sortKey="Fremont, D H" uniqKey="Fremont D">D. H. Fremont</name></author><author><name sortKey="Pierson, T C" uniqKey="Pierson T">T. C. Pierson</name></author><author><name sortKey="Diamond, M S" uniqKey="Diamond M">M. S. Diamond</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huang, K J" uniqKey="Huang K">K. J. Huang</name></author><author><name sortKey="Yang, Y C" uniqKey="Yang Y">Y. C. Yang</name></author><author><name sortKey="Lin, Y S" uniqKey="Lin Y">Y. S. Lin</name></author><author><name sortKey="Huang, J H" uniqKey="Huang J">J. H. Huang</name></author><author><name sortKey="Lin, H S" uniqKey="Lin H">H. S. Lin</name></author><author><name sortKey="Yeh, T M" uniqKey="Yeh T">T. M. Yeh</name></author><author><name sortKey="Chen, S H" uniqKey="Chen S">S. H. Chen</name></author><author><name sortKey="Liu, C C" uniqKey="Liu C">C. C. Liu</name></author><author><name sortKey="Lei, H Y" uniqKey="Lei H">H. Y. Lei</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="He, Y X" uniqKey="He Y">Y. X. He</name></author><author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name></author><author><name sortKey="Siddiqui, P" uniqKey="Siddiqui P">P. Siddiqui</name></author><author><name sortKey="Niu, J" uniqKey="Niu J">J. Niu</name></author><author><name sortKey="Jiang, S B" uniqKey="Jiang S">S. B. Jiang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="He, Y X" uniqKey="He Y">Y. X. 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="Kou, Z H" uniqKey="Kou Z">Z. H. Kou</name></author><author><name sortKey="Liu, S W" uniqKey="Liu S">S. W. Liu</name></author><author><name sortKey="Jiang, S B" uniqKey="Jiang S">S. B. Jiang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liang, Y F" uniqKey="Liang Y">Y. F. Liang</name></author><author><name sortKey="Wan, Y" uniqKey="Wan Y">Y. Wan</name></author><author><name sortKey="Qiu, Li W" uniqKey="Qiu L">Li. W. Qiu</name></author><author><name sortKey="Zhou, J" uniqKey="Zhou J">J. Zhou</name></author><author><name sortKey="Ni, B" uniqKey="Ni B">B. Ni</name></author><author><name sortKey="Guo, B" uniqKey="Guo B">B. Guo</name></author><author><name sortKey="Zou, Q" uniqKey="Zou Q">Q. Zou</name></author><author><name sortKey="Zou, L Y" uniqKey="Zou L">L. Y. Zou</name></author><author><name sortKey="Zhou, W" uniqKey="Zhou W">W. Zhou</name></author><author><name sortKey="Jia, Z C" uniqKey="Jia Z">Z. C. Jia</name></author><author><name sortKey="Che, X Y" uniqKey="Che X">X. Y. Che</name></author><author><name sortKey="Wu, Y Z" uniqKey="Wu Y">Y. Z. Wu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hu, H" uniqKey="Hu H">H. Hu</name></author><author><name sortKey="Li, L" uniqKey="Li L">L. Li</name></author><author><name sortKey="Kao, R Y" uniqKey="Kao R">R. Y. Kao</name></author><author><name sortKey="Kou, B" uniqKey="Kou B">B. Kou</name></author><author><name sortKey="Wang, Z" uniqKey="Wang Z">Z. Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tam, J P" uniqKey="Tam J">J. P. Tam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qin, C" uniqKey="Qin C">C. Qin</name></author><author><name sortKey="Wang, J" uniqKey="Wang J">J. Wang</name></author><author><name sortKey="Wei, Q" uniqKey="Wei Q">Q. Wei</name></author><author><name sortKey="She, M" uniqKey="She M">M. She</name></author><author><name sortKey="Marasco, W A" uniqKey="Marasco W">W. A. Marasco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wong, S K" uniqKey="Wong S">S. K. Wong</name></author><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Moore, M J" uniqKey="Moore M">M. J. Moore</name></author><author><name sortKey="Choe, H" uniqKey="Choe H">H. Choe</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M. Farzan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Zhang, C" uniqKey="Zhang C">C. Zhang</name></author><author><name sortKey="Sui, J" uniqKey="Sui J">J. Sui</name></author><author><name sortKey="Kuhn, J H" uniqKey="Kuhn J">J. H. Kuhn</name></author><author><name sortKey="Moore, M J" uniqKey="Moore M">M. J. Moore</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xu, Y" uniqKey="Xu Y">Y. Xu</name></author><author><name sortKey="Zhu, J" uniqKey="Zhu J">J. Zhu</name></author><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y. Liu</name></author><author><name sortKey="Lou, Z" uniqKey="Lou Z">Z. Lou</name></author><author><name sortKey="Yuan, F" uniqKey="Yuan F">F. Yuan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xu, Y" uniqKey="Xu Y">Y. Xu</name></author><author><name sortKey="Lou, Z" uniqKey="Lou Z">Z. Lou</name></author><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y. Liu</name></author><author><name sortKey="Pang, H" uniqKey="Pang H">H. Pang</name></author><author><name sortKey="Tien, P" uniqKey="Tien P">P. Tien</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, S F" uniqKey="Wang S">S. F. Wang</name></author><author><name sortKey="Tseng, S P" uniqKey="Tseng S">S. P. Tseng</name></author><author><name sortKey="Yen, C H" uniqKey="Yen C">C. H. Yen</name></author><author><name sortKey="Yang, J Y" uniqKey="Yang J">J. Y. Yang</name></author><author><name sortKey="Tsao, C H" uniqKey="Tsao C">C. H. Tsao</name></author><author><name sortKey="Shen, C W" uniqKey="Shen C">C. W. Shen</name></author><author><name sortKey="Chen, K H" uniqKey="Chen K">K. H. Chen</name></author><author><name sortKey="Liu, F T" uniqKey="Liu F">F. T. Liu</name></author><author><name sortKey="Liu, W T" uniqKey="Liu W">W. T. Liu</name></author><author><name sortKey="Chen, Y M" uniqKey="Chen Y">Y. M. Chen</name></author><author><name sortKey="Huang, J C" uniqKey="Huang J">J. C. Huang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yip, M S" uniqKey="Yip M">M. S. Yip</name></author><author><name sortKey="Leung, N H" uniqKey="Leung N">N. H. Leung</name></author><author><name sortKey="Cheung, C Y" uniqKey="Cheung C">C. Y. Cheung</name></author><author><name sortKey="Li, P H" uniqKey="Li P">P. H. Li</name></author><author><name sortKey="Lee, H H" uniqKey="Lee H">H. H. Lee</name></author><author><name sortKey="Daeron, M" uniqKey="Daeron M">M. Daëron</name></author><author><name sortKey="Peiris, J S" uniqKey="Peiris J">J. S. Peiris</name></author><author><name sortKey="Bruzzone, R" uniqKey="Bruzzone R">R. Bruzzone</name></author><author><name sortKey="Jaume, M" uniqKey="Jaume M">M. Jaume</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jaume, M" uniqKey="Jaume M">M. Jaume</name></author><author><name sortKey="Yip, M S" uniqKey="Yip M">M. S. Yip</name></author><author><name sortKey="Cheung, C Y" uniqKey="Cheung C">C. Y. Cheung</name></author><author><name sortKey="Leung, H L" uniqKey="Leung H">H. L. Leung</name></author><author><name sortKey="Li, P H" uniqKey="Li P">P. H. Li</name></author><author><name sortKey="Kien, F" uniqKey="Kien F">F. Kien</name></author><author><name sortKey="Dutry, I" uniqKey="Dutry I">I. Dutry</name></author><author><name sortKey="Callendret, B" uniqKey="Callendret B">B. Callendret</name></author><author><name sortKey="Escriou, N" uniqKey="Escriou N">N. Escriou</name></author><author><name sortKey="Altmeyer, R" uniqKey="Altmeyer R">R. Altmeyer</name></author><author><name sortKey="Nal, B" uniqKey="Nal B">B. Nal</name></author><author><name sortKey="Daeron, M" uniqKey="Daeron M">M. Daëron</name></author><author><name sortKey="Bruzzone, R" uniqKey="Bruzzone R">R. Bruzzone</name></author><author><name sortKey="Peiris, J S" uniqKey="Peiris J">J. S. Peiris</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dawson, P E" uniqKey="Dawson P">P. E. Dawson</name></author><author><name sortKey="Muir, T W" uniqKey="Muir T">T. W. Muir</name></author><author><name sortKey="Clark Lewis, I" uniqKey="Clark Lewis I">I. Clark-Lewis</name></author><author><name sortKey="Kent, S B" uniqKey="Kent S">S. B. Kent</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruan, Y J" uniqKey="Ruan Y">Y. J. Ruan</name></author><author><name sortKey="Wei, C L" uniqKey="Wei C">C. L. Wei</name></author><author><name sortKey="Ee, A L" uniqKey="Ee A">A. L. Ee</name></author><author><name sortKey="Vega, V B" uniqKey="Vega V">V. B. Vega</name></author><author><name sortKey="Thoreau, H" uniqKey="Thoreau H">H. Thoreau</name></author><author><name sortKey="Su, S T" uniqKey="Su S">S. T. Su</name></author><author><name sortKey="Chia, J M" uniqKey="Chia J">J. M. Chia</name></author><author><name sortKey="Ng, P" uniqKey="Ng P">P. Ng</name></author><author><name sortKey="Chiu, K P" uniqKey="Chiu K">K. P. Chiu</name></author><author><name sortKey="Lim, L" uniqKey="Lim L">L. Lim</name></author><author><name sortKey="Zhang, T" uniqKey="Zhang T">T. Zhang</name></author><author><name sortKey="Peng, C K" uniqKey="Peng C">C. K. Peng</name></author><author><name sortKey="Lin, E O" uniqKey="Lin E">E. O. Lin</name></author><author><name sortKey="Lee, N M" uniqKey="Lee N">N. M. Lee</name></author><author><name sortKey="Yee, S L" uniqKey="Yee S">S. L. Yee</name></author><author><name sortKey="Ng, L F" uniqKey="Ng L">L. F. Ng</name></author><author><name sortKey="Chee, R E" uniqKey="Chee R">R. E. Chee</name></author><author><name sortKey="Stanton, L W" uniqKey="Stanton L">L. W. Stanton</name></author><author><name sortKey="Long, P M" uniqKey="Long P">P. M. Long</name></author><author><name sortKey="Liu, E T" uniqKey="Liu E">E. T. Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sakaguchi, N" uniqKey="Sakaguchi N">N. Sakaguchi</name></author><author><name sortKey="Kimura, T" uniqKey="Kimura T">T. Kimura</name></author><author><name sortKey="Matsushita, S" uniqKey="Matsushita S">S. Matsushita</name></author><author><name sortKey="Fujimura, S" uniqKey="Fujimura S">S. Fujimura</name></author><author><name sortKey="Shibata, J" uniqKey="Shibata J">J. Shibata</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuwahara, K" uniqKey="Kuwahara K">K. Kuwahara</name></author><author><name sortKey="Yoshida, M" uniqKey="Yoshida M">M. Yoshida</name></author><author><name sortKey="Kondo, E" uniqKey="Kondo E">E. Kondo</name></author><author><name sortKey="Sakata, A" uniqKey="Sakata A">A. Sakata</name></author><author><name sortKey="Watanabe, Y" uniqKey="Watanabe Y">Y. Watanabe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jonsson, U" uniqKey="Jonsson U">U. Jönsson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Johnsson, B" uniqKey="Johnsson B">B. Johnsson</name></author><author><name sortKey="Lofas, S" uniqKey="Lofas S">S. Löfas</name></author><author><name sortKey="Lindquist, G" uniqKey="Lindquist G">G. Lindquist</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Karlsson, R" uniqKey="Karlsson R">R. Karlsson</name></author><author><name sortKey="Michaelsson, A" uniqKey="Michaelsson A">A. Michaelsson</name></author><author><name sortKey="Mattsson, L" uniqKey="Mattsson L">L. Mattsson</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article" xml:lang="EN"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">ACS Infect Dis</journal-id><journal-id journal-id-type="iso-abbrev">ACS Infect Dis</journal-id><journal-id journal-id-type="publisher-id">id</journal-id><journal-id journal-id-type="coden">aidcbc</journal-id><journal-title-group><journal-title>ACS Infectious Diseases</journal-title></journal-title-group><issn pub-type="epub">2373-8227</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27627203</article-id><article-id pub-id-type="pmc">7075522</article-id><article-id pub-id-type="doi">10.1021/acsinfecdis.6b00006</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Immunodominant SARS Coronavirus Epitopes in Humans
Elicited both Enhancing and Neutralizing Effects on Infection in Non-human
Primates</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Wang</surname><given-names>Qidi</given-names></name><xref rid="aff2" ref-type="aff">‡</xref><xref rid="notes-2" ref-type="notes">∥</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Zhang</surname><given-names>Lianfeng</given-names></name><xref rid="aff3" ref-type="aff">§</xref><xref rid="notes-2" ref-type="notes">∥</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Kuwahara</surname><given-names>Kazuhiko</given-names></name><xref rid="aff4" ref-type="aff">Δ</xref><xref rid="notes-2" ref-type="notes">∥</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Li</surname><given-names>Li</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Liu</surname><given-names>Zijie</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath6"><name><surname>Li</surname><given-names>Taisheng</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath7"><name><surname>Zhu</surname><given-names>Hua</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath8"><name><surname>Liu</surname><given-names>Jiangning</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath9"><name><surname>Xu</surname><given-names>Yanfeng</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath10"><name><surname>Xie</surname><given-names>Jing</given-names></name><xref rid="aff5" ref-type="aff">⊥</xref></contrib><contrib contrib-type="author" id="ath11"><name><surname>Morioka</surname><given-names>Hiroshi</given-names></name><xref rid="aff4" ref-type="aff">Δ</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath12"><name><surname>Sakaguchi</surname><given-names>Nobuo</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff4" ref-type="aff">Δ</xref><xref rid="aff6" ref-type="aff">⊗</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath13"><name><surname>Qin</surname><given-names>Chuan</given-names></name><xref rid="cor2" ref-type="other">*</xref><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath14"><name><surname>Liu</surname><given-names>Gang</given-names></name><xref rid="cor3" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff2" ref-type="aff">‡</xref></contrib><aff id="aff1"><label>†</label>Tsinghua-Peking Center for Life Sciences & School of Pharmaceutical Sciences,<institution>Tsinghua University</institution>, Haidian District, Beijing 100084,<country>People’s Republic of China</country></aff><aff id="aff2"><label>‡</label>Institute of Materia Medica,<institution>Chinese Academy of Medical Sciences & Peking Union Medical College</institution>, 2A Nanwei Road, Xuanwu District, Beijing 100050,<country>People’s Republic of China</country></aff><aff id="aff3"><label>§</label>Institute of Laboratory Animal Science,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100021,<country>People’s Republic of China</country></aff><aff id="aff4"><label>Δ</label>Faculty of Life Sciences,<institution>Kumamoto University</institution>, 1-1-1 Honjo, Kumamoto 860-8556,<country>Japan</country></aff><aff id="aff5"><label>⊥</label>Department of Infectious Disease, Peking Union Medical College Hospital and AIDS Research Center,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, Beijing 100071,<country>People’s Republic of China</country></aff><aff id="aff6"><label>⊗</label>WPI Immunology Frontier Research Center,<institution>Osaka University</institution>, 3-1 Yamada-oka, Suita, Osaka 565-0871,<country>Japan</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>(N.S.) E-mail: <email>nobusaka@ifrec.osaka-u.ac.jp</email>.</corresp><corresp id="cor2"><label>*</label>(C.Q.) E-mail: <email>qinchuan@pumc.edu.cn</email>.</corresp><corresp id="cor3"><label>*</label>(G.L.) E-mail: <email>gangliu27@tsinghua.edu.cn</email>.</corresp></author-notes><pub-date pub-type="epub"><day>30</day><month>03</month><year>2016</year></pub-date><pub-date pub-type="ppub"><day>13</day><month>05</month><year>2016</year></pub-date><volume>2</volume><issue>5</issue><fpage>361</fpage><lpage>376</lpage><history><date date-type="received"><day>07</day><month>01</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016 American Chemical
Society</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>American Chemical
Society</copyright-holder><license license-type="open-access"><license-p>This article is made available via the PMC Open Access Subset 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 the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="id6b00006_0011" id="ab-tgr1"></graphic></p><p>Severe
acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV)
and has the potential to threaten global public health and socioeconomic
stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV
infection in vitro and in non-human primates clouds the prospects
for a safe vaccine. Using antibodies from SARS patients, we identified
and characterized SARS-CoV B-cell peptide epitopes with disparate
functions. In rhesus macaques, the spike glycoprotein peptides S<sub>471–503</sub>, S<sub>604–625</sub>, and S<sub>1164–1191</sub> elicited antibodies that efficiently prevented infection in non-human
primates. In contrast, peptide S<sub>597–603</sub> induced
antibodies that enhanced infection both in vitro and in non-human
primates by using an epitope sequence-dependent (ESD) mechanism. This
peptide exhibited a high level of serological reactivity (64%), which
resulted from the additive responses of two tandem epitopes (S<sub>597–603</sub> and S<sub>604–625</sub>) and a long-term
human B-cell memory response with antisera from convalescent SARS
patients. Thus, peptide-based vaccines against SARS-CoV could be engineered
to avoid ADE via elimination of the S<sub>597–603</sub> epitope.
We provide herein an alternative strategy to prepare a safe and effective
vaccine for ADE of viral infection by identifying and eliminating
epitope sequence-dependent enhancement of viral infection.</p></abstract><kwd-group><kwd>SARS-CoV</kwd><kwd>peptide</kwd><kwd>vaccine</kwd><kwd>antibody-dependent enhancement
(ADE)</kwd><kwd>epitope sequence-dependent (ESD) enhancement</kwd><kwd>B-cell peptide epitope</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>id6b00006</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>id6b00006</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id="sec1">A novel severe acute respiratory syndrome coronavirus (SARS-CoV)
was characterized in March 2003 after a global effort following the
first epidemiological case in November 2002.<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> Ten years later (2012), a novel Middle East respiratory syndrome
(MERS) coronavirus emerged, which was a β-coronavirus, similar
to SARS-CoV.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> SARS is a deadly infectious
disease and has the potential to seriously threaten public health
and socioeconomic stability worldwide.<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> Serologic and genetic investigations indicate that SARS-CoV is of
zoonotic origin,<sup><xref ref-type="bibr" rid="ref3">3</xref>,<xref ref-type="bibr" rid="ref4">4</xref></sup> with bats as the likely animal
reservoir. Since the identification of SARS-CoV in 2003, there have
been major advances in understanding SARS-CoV genetics<sup><xref ref-type="bibr" rid="ref1">1</xref>,<xref ref-type="bibr" rid="ref5">5</xref>,<xref ref-type="bibr" rid="ref6">6</xref></sup> and molecular epidemiology<sup><xref ref-type="bibr" rid="ref7">7</xref></sup> and in identification of host receptors<sup><xref ref-type="bibr" rid="ref8">8</xref>,<xref ref-type="bibr" rid="ref9">9</xref></sup> and T-cell epitopes.<sup><xref ref-type="bibr" rid="ref10">10</xref>,<xref ref-type="bibr" rid="ref11">11</xref></sup> Models in mice, ferrets, and
monkeys have been established, each with particular strengths and
weaknesses.<sup><xref ref-type="bibr" rid="ref12">12</xref></sup> Vaccine candidates have included
DNA, a live attenuated strain, recombinant proteins, inactivated whole
virions, and vector vaccines;<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> however,
none have been approved through clinical investigation.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Safety concerns about a SARS-CoV vaccine have
been raised, given the observation in vitro of antibody-dependent
enhancement (ADE) of SARS-CoV infection that could, in theory, exacerbate
disease.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> Other observations include evidence
of ADE reported here for the first time induced by an inactivated
SARS-CoV vaccine in rhesus macaques (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>) and by antisera from SARS patients (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S1</ext-link>), as well as ADE in other coronavirus
infections.<sup><xref ref-type="bibr" rid="ref16">16</xref>−<xref ref-type="bibr" rid="ref18">18</xref></sup></p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Observation of ADE induced by inactivated SARS-CoV vaccine
in the lungs (H&E staining, 200×). The monkeys were immunized
intramuscularly with formalin-inactivated SARS-CoV virions and boosted
on day 14 with the same dose of 1.0 mL/monkey (4 × 10<sup>4</sup> TCID<sub>50</sub>). The animals were then challenged with nasal
cavity inoculation of SARS-CoV (1 × 10<sup>6</sup> TCID<sub>50</sub> in 4.0 mL/monkey, PUMC01 SARS-CoV strain) 14 days after the boost.
The animals were sacrificed 6 days after viral challenge, and lung
tissues were sampled for general and pathological observations. Pathological
examination procedures were as described in ref (<xref ref-type="bibr" rid="ref37">37</xref>). Infected macaque: lung
interval broadened, visible macrophage infiltration with alveolar
epithelial hyperplasia. ADE macaque: lung interval broadened, lung
interval fractured with large amounts of macrophage and lymphocyte
infiltration, visible fibrin and protein-rich edema in alveolar cavity.
Protected macaques: lung interval slightly broadened, without visible
abnormalities. The arrows indicate the lung lesions of infected and
ADE animals.</p></caption><graphic xlink:href="id6b00006_0001" id="gr2" position="float"></graphic></fig><p>ADE in vitro has been
observed for many other viruses, including yellow fever virus, West
Nile virus, human immunodeficiency virus (HIV), Ebola virus, respiratory
syncytial virus (RSV), and influenza A virus.<sup><xref ref-type="bibr" rid="ref19">19</xref>−<xref ref-type="bibr" rid="ref21">21</xref></sup> HIV-induced
ADE has been described,<sup><xref ref-type="bibr" rid="ref22">22</xref>,<xref ref-type="bibr" rid="ref23">23</xref></sup> but the exact mechanism
is still uncertain.<sup><xref ref-type="bibr" rid="ref24">24</xref></sup> Among other examples
of ADE, secondary infection with dengue virus of a heterologous serotype
has been associated with an immunopathologic vascular leakage and
hemorrhagic syndrome, Dengue hemorrhagic fever/dengue shock syndrome
(DHF/DSS).<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> Most descriptions of ADE relate
to Fc-gamma receptor (FcγR) and/or complement components promoting
viral uptake and virus replication pathways.<sup><xref ref-type="bibr" rid="ref26">26</xref>−<xref ref-type="bibr" rid="ref28">28</xref></sup> Goncalvez et
al. reported that treatment of juvenile rhesus monkeys with the cross-reactive
mAb 1A5, which recognizes the fusion loop in DII of Dengue virus,
enhanced DV4 viremia by several logs within 3–6 days of infection.<sup><xref ref-type="bibr" rid="ref26">26</xref></sup> Furthermore, a 9-amino acid (aa) deletion at
the N-terminus of the CH<sub>2</sub> domain in the Fc region abrogated
enhancement in vitro and in vivo, confirming that ADE occurred through
an FcγR pathway for this class of antibodies. Another study
analyzed the stoichiometric relationship between antibody-mediated
neutralization and enhancement of West Nile virus (WNV) infection
in cells expressing FcγR.<sup><xref ref-type="bibr" rid="ref29">29</xref></sup> The results
showed that there is an antibody occupancy threshold on the virion
for neutralization or enhancement of virus infection. Strongly neutralizing
DIII-lateral ridge-specific mAbs inhibit at lower occupancy, whereas
weakly neutralizing mAbs that bind distinct epitopes require a much
higher mAb occupancy for neutralization. When mAb occupancy falls
below the threshold for neutralization, ADE can occur. In subsequent
studies, the same group showed that the level of antibody occupancy
that promotes ADE in vivo is also modulated by the binding of C1q,
which restricts ADE in an IgG subclass-specific manner.<sup><xref ref-type="bibr" rid="ref30">30</xref></sup> On the basis of these studies, many neutralizing
antibodies may enhance infection in vitro in FcγR<sup>+</sup> cells when their concentrations fall below a key occupancy threshold,
and some Abs that poorly neutralize may strongly enhance over a wide
dose–response range. However, for antibody isotypes that bind
C1q avidly, much of the enhancing effect may be minimized in vivo.</p><p>Other studies suggest that FcγR-dependent ADE may not be
the only mechanism for antibody enhancement of infection. Huang et
al.<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> demonstrated ADE in vitro of an anti-prM
mAb against DENV in cell lines that lacked expression of FcγR
(e.g., baby hamster kidney cell line BHK-21 and murine fibroblast
cell lineNIH3T3). Importantly, a peptide in the M3 region (CPFLKQNEPEDIDCW)
of prM blocked this ADE in a dose-dependent manner. The antibody appeared
to have dual specificity and bound to Dengue virus virions and/or
a cross-reactive HSP60 protein on the cell surface. Thus, ADE via
non-FcγR- or complement-dependent mechanisms is plausible for
DENV, yet poorly characterized in vitro and in vivo. Herein, we discovered
that a peptide of the viral sequence simultaneously elicits the antibodies
of disparate functions in protection and enhancement against SARS-CoV
infection by the studies with host Vero E6 cells in vitro and in non-human
primates.</p><sec id="sec2"><title>Results</title><sec id="sec2.1"><title>Observation of ADE Induced by Inactivated
SARS-CoV Vaccine in the Lungs of Macaques</title><p>First, we observed
the ADE of SARS-CoV infection in lungs after immunization with the
inactivated virus vaccine in macaques. The monkeys with or without
the immunization of inactivated SARS-CoV vaccine were infected by
nasal cavity inoculation of live SARS-CoV virions (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>). Infected macaques showed
lungs with broadened interval and visible macrophage infiltration
with alveolar epithelial hyperplasia. However, ADE macaques after
vaccination showed lungs with broadened interval, lung interval fractured
with large amounts of macrophage and lymphocyte infiltration, visible
fibrin, and protein-rich edema in the alveolar cavity. Protected macaques
showed lungs with slightly broadened intervals without visible abnormalities.
The results implied that the simple vaccination with inactivated whole
SARS-CoV virions might be ineffective for protection of virus infection.</p></sec><sec id="sec2.2"><title>Identification of Highly Immunodominant B-Cell Peptides of SARS-CoV
in Humans</title><p>Mapping highly immunoreactive human B-cell peptides
by employing SARS patient antisera was carried out by a number of
research groups. Consistent with previous reports, Jiang’s
laboratory reported that the M1–131 and M132–162 peptides<sup><xref ref-type="bibr" rid="ref32">32</xref></sup> from the membrane protein and the N153–178
and N362–412 peptides from the N protein<sup><xref ref-type="bibr" rid="ref33">33</xref></sup> were highly reactive with all convalescent-phase test sera
from SARS patients. The laboratories of Wu and Che collaboratively
identified three conformational (amino acids 1–69, 68–213,
and 337–422) and three linear (amino acids 1–69, 121–213,
and 337–422) epitopes on the full-length N protein that were
immunodominant.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> Using a “split
and mix” combinatorial strategy, we synthesized an overlapping
peptide library spanning all four structural proteins of the SARS-CoV
BJ01 strain, including the envelope (E), membrane (M), nucleocapsid
(N), and spike (S). We screened this peptide library with antisera
from convalescent SARS patients and identified dominant peptide epitopes
recognized by anti-SARS IgG.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> Further
experiments in this paper indicate that three peptides in the S protein
(S<sub>471–503</sub>, S<sub>604–625</sub>, and S<sub>1164–1191</sub>) were strongly bound by SARS-CoV-specific
human IgG from 12.8, 32.6, and 13.9%, respectively, of the 470 SARS
patients (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>).
Extension of the peptides (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S2</ext-link>) to
further characterize the epitopes’ N- and C-termini identified
a new immunodominant peptide (S<sub>597–625</sub>), which was
recognized by antisera from 64% of the patients (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>). The S<sub>597–625</sub> peptide specifically reduced the titer of human IgG in convalescent
sera from SARS patients who reacted with SARS-CoV viral lysates (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>A). We studied sequentially
collected sera from 21 convalescent SARS patients whose sera (obtained
3 months after onset of infection) strongly reacted with the S<sub>597–625</sub> peptide. The antisera from these patients continued
to recognize the S<sub>597–625</sub> at 12 months [12/21 (57.1%)],
18 months [7/21 (33.3%)], and 24 months [6/21 (28.6%)] after the onset
of SARS (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>B),
implying that the S<sub>597–625</sub> peptide is a major immunodominant
peptide in humans and elicits a long-term B-cell memory response after
natural infection with SARS-CoV.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>Peptides Recognized by SARS-Infected Patients, Relevant
Mouse mAbs, and Functional Classification<xref rid="t1fn1" ref-type="table-fn">a</xref></title></caption><graphic xlink:href="id6b00006_0010" id="fx1" position="float"></graphic><table-wrap-foot><fn id="t1fn1"><label>a</label><p>*, red-, blue-, and green-colored peptides represent
the epitopes that were assembled into immunogenic peptides. ζ,
neu (neutralizing) or ADE indicates the ability of the individual
mAb to block or enhance, respectively, SARS-CoV infection of Vero
E6 cells. #, the reactivity of individual peptides with antisera from
470 convalescent SARS patients (male, 192; female, 278) was determined
using an ELISA. Commercially available ELISA kits were used to confirm
that 81% of the 470 antisera were positive against viral lysates as
antigen (cutoff value = 0.1). The titer of each serum was averaged
from duplicate wells.</p></fn></table-wrap-foot></table-wrap><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Immunogenic
specificity and long-term memory response of IgG from antisera of
SARS patients to S<sub>597–625</sub> peptide. (A) The reactivity
of human IgG to SARS viral lysates (ELISA kit: Huada S20030004, Beijing,
China) is specifically reduced in a dose-dependent manner by S<sub>597–625</sub>, but not by the hepatitis B virus (HBV) peptide
MDIDPYKEFGATVELLSFLP. P223, P73,
and P194 represent three antisera from convalescent SARS patients.
Reactivity of human IgG with the S<sub>597–625</sub> peptide
was determined by an ELISA. Lysates (0.025 μg/well) were incubated
overnight at room temperature and then blocked by 5% goat serum in
PBS (125 μL) for 2 h. Ten microliters of antiserum and 100 μL
of PBS were further incubated for 30 min at 37 °C. Proper amounts
of goat anti-human IgG conjugated to HRP were used for detection of
OD<sub>450</sub> values. Each antiserum was used in duplicate, and
the cutoff value was 0.1. (B) The antisera of 21 convalescent SARS
patients were collected and tested 3 (M3), 12 (M12), 18 (M18), and
24 (M24) months after onset of SARS-CoV infection. The antiserum of
patient 9 collected 3 months after onset of infection was omitted
due to insufficient sample quantity. The peptides were dissolved in
a minimal volume of DMSO and then diluted to a final concentration
of 10 μg/mL in carbonate buffer (pH 9.6). In total, 1.0 μg/well
was used for capture antibodies from antiserum. The detailed procedure
was the same as in (A) with a cutoff value of 0.1.</p></caption><graphic xlink:href="id6b00006_0002" id="gr3" position="float"></graphic></fig></sec><sec id="sec2.3"><title>Epitope-Specific Antibodies
of SARS-CoV Exhibited Enhancing or Neutralizing Functions in Vitro</title><p>To study the functional significance of immunodominant peptides
of the S glycoprotein, we generated 23 monoclonal antibodies (mAbs, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S3</ext-link>) by immunization of mice. Ten of these
strongly bound to the SARS-CoV virion (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>A). Among them, five blocked SARS-CoV infection
of Vero E6 cells (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>B), including mAb4E5, mAb4A10, and mAb6G5 against S<sub>471–503</sub>, mAb11B1 against S<sub>604–625</sub>, and mAb126–10
against S<sub>1164–1191</sub>.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Generated monoclonal antibodies bound
to SARS-CoV virions and shared disparate functions. (A) The defined
monoclonal antibodies bound to SARS-CoV virions of SARS-CoV PUMC01
strain (4 × 10<sup>–5</sup> TCID<sub>50</sub>/mL).<sup><xref ref-type="bibr" rid="ref46">46</xref></sup> The cutoff value was set to 0.1 (the average
for binding of unrelated mAb-HIV-P27). (B) Neutralization or enhancement
of the SARS-CoV infection in Vero E6 cells by mAbs (1.0 μg/mL)
was specifically reduced by the corresponding immunopeptide (0.1 μg/mL),
but not by a hepatitis B virus (HBV) peptide (0.1 μg/mL). HBV
peptide (LLDYQGMLPV) is an unrelated control peptide from
an HBV surface protein. S<sub>597–625</sub> and HBV peptide
were pre-incubated with the corresponding mAbs or human antisera for
30 min at 4 °C before function was tested. These experiments
were performed in triplicate, and the data are presented as the mean
± standard deviation.</p></caption><graphic xlink:href="id6b00006_0003" id="gr4" position="float"></graphic></fig><p>In contrast, two mAbs (mAb43-3-14 and mAb219-10-28) against
S<sub>597–625</sub>, despite sharing some of the same isotypes
and constant region configurations with neutralizing antipeptide antibodies
(IgG1/κ, IgG2a/κ, and IgG2b/κ; <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>), markedly enhanced SARS-CoV
infection of Vero E6 cells (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>B). This was particularly striking given that Vero
E6 cells lack FcγR, and the best-understood mechanism for ADE
involves Fc-mediated internalization and replication of virus. mAb43-3-14-
and mAb219-10-28-enhanced Vero E6 cell infection was reduced in a
dose-dependent manner by S<sub>597–625</sub>, but not by an
unrelated HBV peptide (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>A,B), whereas ADE of human antisera from SARS-CoV-infected
patients was also specifically blocked by the same peptide (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>C,D), implying that
peptide-based ADE occurred during the epidemic period of a SARS outbreak.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Antibody-dependent
enhancement of SARS-CoV infection. (A–D) Enhancement of SARS-CoV
infection by either mAbs (1.0 μg/mL) or human antisera (1:500)
was reduced in a peptide dose-dependent manner. hW49 and hS101 represent
two antisera from convalescent SARS patients 3 months after onset.
HBV peptide (LLDYQGMLPV) is an unrelated control peptide
from HBV surface protein. S<sub>597–625</sub> and HBV peptide
were pre-incubated with the corresponding mAb or human antisera for
30 min at 4 °C before function was tested. (E) Alanine scanning
mutagenesis of the S<sub>597–606</sub> showed the minimum requirement
(S<sub>597–603</sub>) for N-terminal binding of S<sub>597–625</sub> to mAb43-3-14. This experiment was performed in triplicate, and
the data are presented as the mean ± standard deviation.</p></caption><graphic xlink:href="id6b00006_0004" id="gr5" position="float"></graphic></fig><p>An antibody-peptide binding ELISA
indicated that mAb11B1 against S<sub>604–625</sub> reacts with
both S<sub>597–625</sub> and S<sub>604–625</sub>, whereas
mAb43-3-14 against S<sub>597–625</sub> only bound to S<sub>597–625</sub> (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>E). The data suggest that mAb11B1 was induced by S<sub>604–625</sub> and is a consensus peptide of S<sub>597–625</sub> and S<sub>604–625</sub>, whereas mAb43-3-14 was from the N-terminal
peptide S<sub>597–603</sub> (LYQDVNC) immunizing antigen.
Synthesized peptides LYQDVN (S<sub>597–602</sub>), LYQDVNC
(S<sub>597–603</sub>), and LYQDVNCT (S<sub>597–604</sub>) were all immunoreactive with the tested sera of 24 SARS-positive
patients, further confirming that the S<sub>597–603</sub> peptide
is an immunodominant one in humans (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Figure S1</ext-link>). Alanine scanning mutagenesis of the S<sub>597–604</sub> peptide demonstrated that L597, Y598, Q599, D600, and C603 are critical
amino acid residues for interaction with mAb43-3-14 (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>E; <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S4</ext-link>). The S<sub>597–603</sub> peptide lies close to the
C-terminus of the SARS-CoV major receptor (ACE2)-binding domain (RBD).<sup><xref ref-type="bibr" rid="ref8">8</xref></sup> We speculate that mAb43-3-14 may expose specific
conformations that catalyze SARS-CoV attachment to and/or membrane
fusion with target cells.</p></sec><sec id="sec2.4"><title>Neutralization or Enhancement of SARS-CoV
Infection in Non-human Primates by Epitope-Specific Antibodies</title><p>Low-molecular-weight peptides behave like haptens, with relatively
low immunogenicity. To make them more immunogenic, we chemically ligated
four copies of each peptide to a lysine core [BrCH<sub>2</sub>CO-Lys<sub>2</sub>-(Lys)-β-Ala-CONH<sub>2</sub>(BrK<sub>2</sub>KA), <xref rid="sch1" ref-type="scheme">Scheme <xref rid="sch1" ref-type="scheme">1</xref></xref>] to prepare a multiple
antigen peptide system (MAP)<sup><xref ref-type="bibr" rid="ref36">36</xref></sup> and purified
them by preparative HPLC (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>A; <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S7</ext-link>). Molecular
weight was confirmed by LC-MS/MS (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref></xref>B).</p><fig id="sch1" position="float"><label>Scheme 1</label><caption><title>Synthesis of Multiple Antigen Peptide
Systems (MAPS) by Chemical Ligation of a Thiol Nucleophilic Substitution
Reaction</title><p id="sch1-fn1">B-cell epitopes with a free
−SH sequence (containing a Cys residue) were prepared directly.
B-cell epitopes without a free −SH group were anchored by an
additional cysteine residue at the C-terminal (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Figure S2</ext-link>). A four-branched, brominated, multiple-antigen
core peptide (BrK<sub>2</sub>KA, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Figure S3</ext-link>) via lysine two α-amino and side-chain amino groups was prepared
that finally conjugated with four copies of individual antigenic peptide.</p></caption><graphic xlink:href="id6b00006_0009" id="gr1" position="float"></graphic></fig><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Tetrameric forms of peptides were successfully synthesized
on a branched lysine scaffold. MAP-S<sub>471–503</sub> = [(S<sub>471–503</sub>)<sub>4</sub>Lys]<sub>2</sub>Lys-β-Ala-CONH<sub>2</sub>; MAP-S<sub>604–625</sub> = [(S<sub>604–625</sub>)<sub>4</sub>Lys]<sub>2</sub>Lys-β-Ala-CONH<sub>2</sub>; MAP-S<sub>597–625</sub> = [(S<sub>597–625</sub>)<sub>4</sub>Lys]<sub>2</sub> Lys-β-Ala-CONH<sub>2</sub>; MAP-S<sub>1164–1191</sub> = [(S<sub>1164–1191</sub>)<sub>4</sub>Lys]<sub>2</sub>Lys-β-Ala-CONH<sub>2</sub>. RP-HPLC profiles (A) of the MAPs were analyzed by a Shimadzu
LC-10AT analytical HPLC system with a C8 column (4.6 mm × 250
mm, 5 μm) and UV detection at 214 nm. The molecular weights
(B) were determined by high-resolution LC-MS in an Agilent LC/MSD
TOF system and hypermass reconstruction of the raw MS data to a single
charge.</p></caption><graphic xlink:href="id6b00006_0005" id="gr6" position="float"></graphic></fig><p>We used these synthetic MAPs to
vaccinate rhesus monkeys (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S4</ext-link>) as
follows: Vac1, control group (received 0.9% NaCl) of four macaques
randomly assigned to two groups for day 2 or day 6 postinfection sacrifice;
Vac2, MAP-S<sub>597–625</sub> given to six macaques randomly
assigned to two groups for day 2 or day 6 postinfection sacrifice;
Vac3, a mixture of MAP-S<sub>471–503</sub>, MAP-S<sub>604–625</sub>, and MAP-S<sub>1164–1191</sub> given to six macaques randomly
assigned to two groups for day 2 or day 6 postinfection sacrifice;
and Vac4, a mixture of MAP-S<sub>471–503</sub>, MAP-S<sub>597–625</sub>, and MAP-S<sub>1164–1191</sub> given to six macaques randomly
assigned to two groups for day 2 or day 6 postinfection sacrifice.
The immunization protocol included one priming injection followed
by three boosts at 2-week intervals (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Chart S1</ext-link>). Antipeptide polyclonal Abs (monkey IgG) monitored by ELISA showed
high-level responses to all immunized peptides after three boosts
(<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>, left).</p><fig id="fig6" position="float"><label>Figure 6</label><caption><p>Rhesus
monkeys strongly responded to peptide-based vaccines (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S5</ext-link>). Vaccinations (A) were designed as
Vac1 (0.9% NaCl) for the control group of four animals, Vac2 (MAPs-S<sub>597–625</sub>) for the enhancement of SARS-CoV infection in
six experimental animals, Vac3 (MAPs-S<sub>471–503</sub>, MAPs-S<sub>604–625</sub>, and MAPs-S<sub>1164–1191</sub>) for
the neutralization of SARS-CoV infection in six experimental animals,
and Vac4 (MAPs-S<sub>471–503</sub>, MAPs-S<sub>597–625</sub>, and MAPs-S<sub>1164–1191</sub>) for the reduction of neutralizing
ability in six experimental animals (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S4</ext-link>). The average IgG levels against peptides in each vaccine group
were monitored by ELISA on the day before injection or boost. IgG
level (B) against S<sub>604–625</sub> is an average of grouped
macaques. The titer of anti-S<sub>604–625</sub> IgG was greater
than 1:10<sup>6</sup> in all relevant immunized monkey groups.</p></caption><graphic xlink:href="id6b00006_0006" id="gr7" position="float"></graphic></fig><p>Antibodies (<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>A) against the four peptides in sera from
Vac1-immunized animals were at background level on all blood collection
days. Vac2 gradually induced anti-S<sub>597–625</sub> peptide
antibodies after three boosts. Not surprisingly, both Vac2 and Vac4
immunizations elicited high levels of IgG against the S<sub>604–625</sub> peptide because the vaccine components consisted of the S<sub>597–625</sub> or S<sub>604–625</sub> epitopes in these two animal groups.
Anti-S<sub>604–625</sub> and -S<sub>1164–1191</sub> antibodies
in the Vac3-immunized animal group reached their highest levels before
the second boost (28 days after the first injection). The average
titer of anti-S<sub>604–625</sub> IgG was greater than 1:10<sup>6</sup> in Vac3-immunized monkeys before SARS-CoV challenge and was
3- or 2-fold times that of Vac2 or Vac4, respectively. Interestingly,
when Vac3-vaccinated animals were challenged by SARS-CoV, the IgG
level against S<sub>604–625</sub> gradually decreased (<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>B) on days 2 and
6 postinfection. This is different from the Vac2 and Vac4 immunizations,
after which IgG levels inexplicably increased on day 2 postinfection
and subsequently decreased on day 6 postinfection. This observation
implies that anti-S<sub>604–625</sub> antibodies are strongly
involved in the neutralization of SARS-CoV in monkeys and that the
existing S<sub>597–603</sub> epitope (in Vac2 and Vac4) may
reduce the total titer of anti-S<sub>604–625</sub> antibodies.</p><p>All immunized macaques were subsequently challenged with SARS-CoV
(PUMC01 strain) via nasal inhalation on the 14th day after the last
boost, as described.<sup><xref ref-type="bibr" rid="ref37">37</xref></sup> At 2 and 6 days
postinfection (DPI), the macaques were sacrificed (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Chart S1; Table S7</ext-link>). The data for lung damage and viral burden
in these macaques are summarized in <xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref></xref>.</p><table-wrap id="tbl2" position="float"><label>Table 2</label><caption><title>Summary of Lung Damage
and Viral Burden of Vaccinated Macaques Challenged by SARS-CoV</title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="char" char="±"></col><col align="char" char="±"></col><col align="char" char="."></col><col align="char" char="."></col></colgroup><thead><tr><th style="border:none;" align="center"> </th><th colspan="2" align="center">gross
lung pathology<xref rid="t2fn1" ref-type="table-fn">a</xref><hr></hr></th><th colspan="2" align="center" char="±">infected cells in lung<xref rid="t2fn2" ref-type="table-fn">b</xref><hr></hr></th><th colspan="2" align="center" char=".">viral burden<xref rid="t2fn3" ref-type="table-fn">c</xref><hr></hr></th></tr><tr><th style="border:none;" align="center">Vac</th><th style="border:none;" align="center">day 2</th><th style="border:none;" align="center">day 6</th><th style="border:none;" align="center" char="±">day 2</th><th style="border:none;" align="center" char="±">day 6</th><th style="border:none;" align="center" char=".">day 2</th><th style="border:none;" align="center" char=".">day 6</th></tr></thead><tbody><tr><td style="border:none;" align="left">1</td><td style="border:none;" align="left">grade IV</td><td style="border:none;" align="left">grade IV</td><td style="border:none;" align="char" char="±">13.5 ± 2.6</td><td style="border:none;" align="char" char="±">10.0 ± 3.0</td><td style="border:none;" align="char" char=".">141,000</td><td style="border:none;" align="char" char=".">136,000</td></tr><tr><td style="border:none;" align="left">2</td><td style="border:none;" align="left">grade IV</td><td style="border:none;" align="left">grade IV</td><td style="border:none;" align="char" char="±">13.8 ± 2.1</td><td style="border:none;" align="char" char="±">9.8 ± 3.0</td><td style="border:none;" align="char" char=".">116,000</td><td style="border:none;" align="char" char=".">143,000</td></tr><tr><td style="border:none;" align="left">3</td><td style="border:none;" align="left">grade I–II</td><td style="border:none;" align="left">grade I–II</td><td style="border:none;" align="char" char="±">7.4 ± 3.2</td><td style="border:none;" align="char" char="±">7.0 ± 2.9</td><td style="border:none;" align="char" char=".">6,200</td><td style="border:none;" align="char" char=".">7,300</td></tr><tr><td style="border:none;" align="left">4</td><td style="border:none;" align="left">grade II–III</td><td style="border:none;" align="left">grade II–III</td><td style="border:none;" align="char" char="±">8.14 ± 3.32</td><td style="border:none;" align="char" char="±">7.8 ± 2.91</td><td style="border:none;" align="char" char=".">6,835</td><td style="border:none;" align="char" char=".">31,254</td></tr></tbody></table><table-wrap-foot><fn id="t2fn1"><label>a</label><p>Average gross lung
pathology was determined by a standard in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S8</ext-link>.</p></fn><fn id="t2fn2"><label>b</label><p>SARS-CoV-infected
macrophages and alveolar epithelial cells in the vaccine groups were
counted by using antipeptide antibodies.</p></fn><fn id="t2fn3"><label>c</label><p>Quantitative viral burden analysis by quantitative RT-PCR
(viral copies/mg lung tissue).</p></fn></table-wrap-foot></table-wrap><p>Gross lung pathology was evident at both 2 and 6 DPI in all groups.
In mock-immunized macaques (Vac1), the average pathology grade was
grade IV (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>A,B; <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S5</ext-link>), with severe diffuse alveolar damage,
including alveolar shrinking and rupture of elastic fibers. Massive
macrophage infiltration was associated with fusion of alveolar septa.
Pulmonary edema, fibrin, hemorrhage, and cellular debris contributed
to the severe interstitial pneumonitis. Damage was similar (grade
IV) for Vac2, somewhat reduced (grade II–III) for Vac4, and
markedly diminished (grade I–II) for Vac3. Immunohistochemistry
using mAbs against the viral peptides revealed a large number of SARS-CoV-infected
macrophages and alveolar epithelial cells in the Vac1 group (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>C), averaging 13.5
± 2.6 infected cells per 10 high-power fields (HPF) and 10.0
± 3.0 infected cells per 10 HPF at 2 and 6 DPI, respectively.
Quantitative viral burden analysis by quantitative RT-PCR showed an
average of 141,000 copies/mg lung tissue (2 DPI) and 136,000 copies/mg
lung tissue (6 DPI) of SARS-CoV in the lungs of the Vac1 group.</p><fig id="fig7" position="float"><label>Figure 7</label><caption><p>Peptide-based
vaccines neutralized or enhanced SARS-CoV infection of rhesus monkeys.
(A) Pathologic changes in lung tissue. The arrows point to the areas
that showed pathology. (B) Histopathologic examination of macaque
lung tissue. Lung damage was pathologically characterized as an average
standard grade (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S5</ext-link>). (C) Immunohistochemical
staining of SARS-CoV-infected cells in lung tissue. Lung tissue sections
were incubated with a mixture of mAb4E5, mAb11B1, and mAb9A6 (each
at 0.01 μg/mL) and developed using horseradish peroxidase (HRP)-labeled
goat anti-mouse IgG (1:1000 dilution, ZhongShan Inc., Guangzhou, PRC).
SARS-CoV-positive cells show brown staining, which localizes in the
cytoplasm of monocytes and pneumocytes (magnification 200×).
Arrows indicate the lung lesions of animals.</p></caption><graphic xlink:href="id6b00006_0007" id="gr8" position="float"></graphic></fig><p>Compared with the Vac1 group, the Vac2 group showed similar
histology at 2 DPI, but the interstitial pneumonia was far worse at
6 DPI, characterized by massive numbers of inflammatory cells in hemorrhagic
septa, with extensive exudation. SARS-CoV-infected cells in the lungs
averaged 13.8 ± 2.1 per 10 HPF at 2 DPI and 9.8 ± 3.0 per
10 HPF at 6 DPI, whereas viral copies were 116,000/mg lung tissue
and 143,000/mg lung tissue, respectively. Thus, even though S<sub>597–625</sub> was immunodominant in patients, antigenic in
monkeys, and reactive with the neutralizing mAb 11B1, it was nonprotective
and even harmful when used as a vaccine.</p><p>In contrast, the immunized
monkeys in the Vac3 group had a strongly increased ability to control
SARS-CoV infection in association with induction of high levels of
anti-S<sub>604–625</sub> antibodies (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref></xref>E). At 2 DPI, hemorrhage in septa and elastic
fibers of the alveolar wall were indicated by apparent inflammation
in silver staining results. Alveolus septa broadening with increasing
infiltration of inflammatory cells were recorded by 6 DPI, indicating
that only early, acute diffuse alveolar damage occurred. SARS-CoV-infected
lung cells in the Vac3 group averaged 7.4 ± 3.2 per 10 HPF (vs
Vac1 group: <italic>p</italic> < 0.01) and 7.0 ± 2.9 per 10
HPF (vs Vac1 group: <italic>p</italic> < 0.01) at 2 and 6 DPI,
respectively. The SARS-CoV burden averaged 6,200 copies/mg lung tissue
at 2 DPI and 7,300 copies/mg lung tissue at 6 DPI. This represents
a ratio of ∼19 and ∼20 between the number of SARS-CoV
copies in the Vac3 and Vac1 groups at 2 and 6 DPI, respectively.</p><p>For animals that received Vac4, symptoms of acute diffuse alveolar
damage were visible, including fusion of thick septa and ruptured
elastic fibers of the alveoli. The average number of SARS-CoV infected
cells in the lung tissues was 8.14 ± 3.32 per 10 HPF at 2 DPI
(vs Vac1 group: <italic>p</italic> < 0.01) and 7.8 ± 2.91
per 10 HPF at 6 DPI (vs Vac1 group: <italic>p</italic> < 0.01).
The SARS-CoV burden averaged 31,254 copies/mg lung tissue at 6 DPI,
that is, 4.5 times the burden of 6,835 copies/mg lung tissue at 2
DPI. The Vac4 group also showed a 4.3-fold increase to Vac3 group
(31,254 copies/mg vs 7,300 copies/mg) of viral burden at 6 DPI, suggesting
that the presence of IgG against S<sub>597–603</sub> facilitated
SARS-CoV infection of immunized macaques.</p><p>In a separate experiment
focused on the phenomenon of ADE in macaques, rhesus monkeys were
divided into six groups (<italic>n</italic> = 3 per group, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S9</ext-link>). Three groups were separately sacrificed
at 2 or 6 DPI; each time point included a control group and two groups
that received the enhancing mAb43-3-14 at doses of 0.2 mg/kg or 1.8
mg/kg 1 day prior to challenge with the SARS-CoV PUMC01 strain. Gross
pathologic changes were again recorded in the control group at a grade
IV level (<xref rid="fig8" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>A,B).
Although clear pathologic changes were observed in one of the three
monkeys in the 0.2 mg/kg group, we concluded that previous treatment
with a dose of 0.2 mg/kg mAb43-3-14 did not, on average, significantly
reduce or facilitate SARS-CoV infection. However, macaques treated
with 1.8 mg/kg mAb43-3-14 showed a marked increase in lung lesions.
The lung lesion area in macaque D4-060060 (4.0 × 2.5 cm<sup>2</sup>) at 6 DPI was the largest in all dic experimental groups. At 6 DPI,
all lungs from the 1.8 mg/kg group showed larger areas of necrosis,
severe sheets of septa fusion, necrotic lesions at the hemorrhagic
septa, and massive macrophage infiltration in the alveoli, indicating
that the interstitial pneumonia was much more severe in the mAb43-3-14-treated
group than in the control group (<xref rid="fig8" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>C). There were more SARS-CoV-infected cells
in the lung tissue (12.5 ± 2.3 per 10 HPF at 2 DPI and 13.4 ±
2.6 per 10 HPF at 6 DPI, <italic>p</italic> < 0.01 vs control group).
The average SARS-CoV burden in the lungs was 911,000 copies/mg lung
tissue at 2 DPI and 944,000 copies/mg lung tissue at 6 DPI, a 10-fold
enhancement compared with the control group at 2 DPI and a 14-fold
enhancement at 6 DPI (<xref rid="fig8" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref></xref>D). This result further confirmed that enhancement of the
SARS-CoV infection in macaques was directly related to antibodies
against S<sub>597–603</sub>.</p><fig id="fig8" position="float"><label>Figure 8</label><caption><p>mAb43-3-14 enhances SARS-CoV infection
of rhesus monkeys. (A) Pathologic changes at 6 DPI. (B) Histopathologic
examination of macaque lung tissues. Lung damage was pathologically
characterized as an average standard grade. Control group, grade IV;
0.2 mg/kg group, grade III–IV; 1.8 mg/kg group, grade IV. (C)
Immunohistochemical staining of SARS-CoV-infected cells in lung tissue.
The staining conditions were the same as in <xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref></xref>. (D) SARS-CoV mRNA in infected monkey lung
tissue was quantitatively analyzed from an average of three animals.
The data are presented as the geometric mean ± standard deviation:
(∗) <italic>p</italic> < 0.05; (∗∗) <italic>p</italic> < 0.01 versus control group; (Δ) <italic>p</italic> < 0.05; (ΔΔ) <italic>p</italic> < 0.01 versus
0.2 mg/kg group. Arrows indicate the lung lesions of animals.</p></caption><graphic xlink:href="id6b00006_0008" id="gr9" position="float"></graphic></fig></sec></sec><sec id="sec3"><title>Discussion</title><p>In
this study, we reported for the first time that a SARS-CoV inactivated
vaccine could induce ADE and lung pathology in experimental rhesus
monkeys. Four antigenic peptides (S<sub>471–503</sub>, S<sub>604–625</sub>, S<sub>597–625</sub>, and S<sub>1164–1191</sub>) from the spike protein of SARS-CoV were identified by high cross-reactivity
with a large number of antisera from convalescent SARS patients. Ten
of 23 mAbs generated against these four peptides bound to the SARS-CoV
virion (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>A),
indicating that each peptide is exposed on the SARS-CoV surface. Among
them, five mAbs against S<sub>471–503</sub>, S<sub>604–625</sub>, and S<sub>1164–1191</sub> blocked SARS-CoV infection of
Vero E6 cells (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>B), and two mAbs (mAb43-3-14 and mAb219-10-28) markedly enhanced
SARS-CoV infection of Vero E6 cells. mAb43-3-14 alone significantly
enhanced SARS-CoV infection at the tested high dose of 1.8 mg/kg in
experimental rhesus monkeys, whereas Vac3 (consisting of S<sub>471–503</sub>, S<sub>604–625</sub>, and S<sub>1164–1191</sub> MAPs)
successfully protected animals from SARS-CoV challenge and lung pathology.
Alanine scanning mutagenesis indicated that L597, Y598, Q599, D600,
and/or C603 are critical amino acid residues and that S<sub>597–603</sub> is responsible for mAb43-3-14-induced enhancement, which may catalyze
SARS-CoV attachment and/or membrane fusion with target cells by exposing
specific conformations of the spike protein.</p><p>S<sub>471–503</sub> is located at the virus RBD<sup><xref ref-type="bibr" rid="ref8">8</xref></sup> and can
block SARS-CoV infection of Vero E6 cells in vitro,<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> probably by interfering with high-affinity attachment of
SARS virions to the human ACE2 receptor at residues T487 and N497
of the RBD.<sup><xref ref-type="bibr" rid="ref38">38</xref>,<xref ref-type="bibr" rid="ref39">39</xref></sup> The coronavirus spike proteins
have been characterized as class I fusion proteins containing highly
conserved heptad repeat regions (HR1 and HR2).<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> Thus, interfering with HR1 binding to HR2 has become a
recent target for prevention of viral fusion and entry into target
cells. S<sub>1164–1191</sub> overlaps HR2 (1145–1184
aa) in the SARS-CoV spike glycoprotein. The mechanism by which anti-S<sub>1164–1191</sub> antibodies reduce the efficiency of SARS-CoV
fusion is likely to be associated with an exposed five-turn α-helix
conformation via HR2 binding to HR1 and an extended conformation formed
by residues 1160–1177 and 1178–1184 of three antiparallel
HR2 helices in an oblique orientation surrounding a parallel, trimeric
coiled-coil of three HR1 helices.<sup><xref ref-type="bibr" rid="ref41">41</xref></sup> Binding
of mAb581-39 to the SARS-CoV virion was the strongest among the mAbs
(<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>A), which
implies that S<sub>1164–1191</sub> peptide may act as a major
immunoantigen.</p><p>Severe lung injury occurred in one challenged
rhesus monkey that had been immunized with an inactivated SARS-CoV
vaccine (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>).
Very strong serologic reactivity of S<sub>597–625</sub> with
antisera from 64.4% of convalescent SARS patients was observed; the
response was to two tandem epitopes (S<sub>597–603</sub> and
S<sub>604–625</sub>), but each epitope generated antibodies
with disparate functions regarding neutralization or enhancement of
SARS-CoV infection. Thus, in persons infected by SARS-CoV, enhancing
antibodies and neutralizing antibodies may partly counteract each
other’s functions.</p><p>SARS-CoV’s ADE was also reported
by other research laboratories. Using an HL-CZ human promonocyte cell
line, Chen and Huang found that higher concentrations of antisera
collected from SARS-CoV-infected patients facilitated SARS-CoV infection
and induced higher levels of virus-induced apoptosis. They further
demonstrated that this phenomenon occurred via antispike protein antibodies
that mediated ADE, but not via anti-N protein antibodies.<sup><xref ref-type="bibr" rid="ref42">42</xref></sup> Bruzzone and Jaume’s laboratory showed
that antispike immune serum increased infection of human monocyte-derived
macrophages by replication-competent SARS-CoV as well as by spike-pseudotyped
lentiviral particles (SARS-CoVpp), although they did not clarify whether
this enhancement was through the FcγRII pathway.<sup><xref ref-type="bibr" rid="ref43">43</xref></sup> Jaume et al. reported that a recombinant, full-length
spike protein trimer potentiated infection of human B cell lines,
despite eliciting a neutralizing and protective immune response in
rodents. The study demonstrated that antispike immune serum, while
inhibiting viral entry in a permissive cell line, potentiated infection
of immune cells by SARS-CoV spike-pseudotyped lentiviral particles,
as well as by replication-competent SARS coronavirus. Jaume and collaborators
proposed that ADE of SARS-CoV utilizes a novel cell entry mechanism
into immune cells.<sup><xref ref-type="bibr" rid="ref44">44</xref></sup></p><p>This study demonstrates
for the first time that an antibody (mAb43-3-14) targeting a specific
linear epitope (S<sub>597–603</sub>) of the SARS-CoV spike
protein can mediate enhancement of virus infection both in vitro and
in non-human primates via an epitope sequence-dependent mechanism.
We also demonstrate that the viral peptides that induce protection
from pathogenesis can be identified and distinguished from those inducing
enhancement in primates. These findings reveal a new mechanism of
virus evasion of host defense that potentially provides an alternative
strategy to prepare safe and effective vaccines against ADE of virus
infections.</p></sec><sec id="sec4"><title>Materials and Methods</title><sec id="sec4.1"><title>Peptide Design and Synthesis</title><p>New
peptides from the spike glycoprotein were designed on the basis of
the information gained in the first round of immunopeptide discovery.<sup><xref ref-type="bibr" rid="ref32">32</xref></sup></p><p>Peptides were synthesized manually by
standard Fmoc chemistry protocols on Rink-amide MBHA resin (NovaBiochem,
San Diego, CA, USA; 0.44 mmol/g). All of the <sc>l</sc>-α-Fmoc-protected
amino acids and coupling reagents (DIC and HOBt) were obtained from
GL Biochem (Shanghai, China). All solvents were of analytical grade
and used without further purification. Each amino acid assembly was
completed by using 3 equiv of <sc>l</sc>-α-Fmoc-protected amino
acids and coupling reagents, and the ninhydrin colorimetric test after
each coupling was carried out to ensure no detectable amino remaining.
In the case that the peptide-bearing beads were colorized (positive)
by ninhydrin test, the free amino group was then blocked by reacting
with 15% acetic anhydride in dichloromethane (DCM) (v/v) for 30 min.
Crude peptides without Cys, Met, Arg, and Trp residues were cleaved
using a one-step TFA cleavage method under the following conditions:
TFA (1.9 mL), deionized water (50 μL), and triethyl silane (50
μL). For peptides containing Cys, Met, Arg, or Trp residues,
the following conditions were used: TFA (1.63 mL), thioanisole (0.1
mL), phenol (0.1 g), deionized water (0.1 mL), EDT (50 μL),
and triethylsilane (20 μL). TFA solution was treated with ice-cooled
diethyl ether to precipitate the peptide. The crude peptide was washed
twice by ice-cooled diethyl ether and dried in the N<sub>2</sub> flow
condition.</p><p>In particular, peptide S<sub>471–503</sub> was synthesized on SynPhase PA RAM <sc>d</sc>-series lanterns (loading
8 μmol/lantern, Mimotopes, Raleigh, NC, USA). The lanterns were
treated with 20% piperidine in dimethylformamide (DMF) for 15 min
for deprotection. The general coupling conditions for lanterns utilized
a solution of 80% DMF and 20% DCM, with reagents at the following
concentrations: AA, 120 mM; HOBt, 144 mM; DIC, 120 mM. For each lantern
a minimum of 500 μL of the activated amino acid solution was
required. Alternatively, bromophenol blue (5 μL/mL coupling
solution, 5M) was used as an indicator to monitor the reaction. Simultaneous
side-chain deprotection and cleavage were carried out using 2.5 mL
per lantern of a solution of 82.5% trifluoroacetic acid (TFA)/5% thioanisole/5%
anisole/5% water/2.5% 1,2-ethanedithiol for 2 h.</p><p>All crude peptides
were purified by HPLC to >95% purity on a semipreparative RP C18
column (Zorbax, 300SB-C18, 9.4 × 250 mm. Agilent, Colorado Springs,
CO, USA) eluting at 4 mL/min with a 0–20% CH<sub>3</sub>CN
(buffer B) gradient in water (buffer A) containing 0.1% TFA over 5
min followed by a 20–65% CH<sub>3</sub>CN over 30 min. Pure
peptides were identified by an Shimadzu LC-10AT analytic HPLC system
with a C8 column (4.6 mm × 250 mm, 5 μm) and Agilent LC-MSD
TOF (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Figure S4</ext-link>; S<sub>604–625</sub>, calculated 2505.83 Da, observed 2505.19 Da; S<sub>1164–1191</sub>, calculated 3439.87 Da, observed 3438.70 Da; S<sub>597–625</sub>, calculated 3238.62 Da, observed 3238.54 Da; S<sub>471–503</sub>, calculated 3863.45 Da, observed 3864.20 Da). The molecular weight
of peptides was calculated by a Peptide Molecular Weight Calculator
of Biopeptide Co., Inc. (<uri xlink:href="https://www.biopeptide.com/PepCalc/">https://www.biopeptide.com/PepCalc/</uri>).</p></sec><sec id="sec4.2"><title>Synthesis and Characterization of Multiple Antigen Peptides
(MAPs)<sup><xref ref-type="bibr" rid="ref36">36</xref></sup></title><p>The branched lysine core
(BrCH<sub>2</sub>CO-Lys<sub>2</sub>-(Lys)-β-Ala-CONH<sub>2</sub>) scaffold was synthesized manually by standard Fmoc chemistry protocols
on 0.22 mmol (500 mg) of Rink-amide MBHA resin (NovaBiochem, 0.44
mmol/g) as described above. <italic>N</italic>-α-Fmoc-Lys (Fmoc)
was used at the branch points. The Fmoc-protected amino acids (5.0
equiv) were assembled by using 5.0 equiv of DIC coupling reagent and
HOBt additive. The N-termini of the four branches of the lysine core
were bromoacetylated on the resin with 10 equiv of bromoacetic acid
and DIC in DMF for 2 h. The bromoacetylated lysine core was cleaved
from the resin after treatment with 5% H<sub>2</sub>O/95% TFA for
2 h. It was then precipitated with cold diethyl ether, lyophilized,
and purified by HPLC on a semipreparative RP C18 column (Zorbax, 300SB-C18,
9.4 × 250 mm) eluting at 4 mL/min with a 0–20% CH<sub>3</sub>CN gradient in water containing 0.1% TFA over 5 min followed
by 20–40% CH<sub>3</sub>CN over 20 min. Pure lysine core (25%
yield) was identified by Agilent LC-MSD TOF: calculated (MH<sup>+</sup>) 957.03 Da, observed 957.05 Da.</p><p>MAPs were prepared for immunizations
using a modified chemical ligation protocol.<sup><xref ref-type="bibr" rid="ref45">45</xref></sup> Ligation was achieved by mixing the lysine core and peptide in 500
μL of solvent at certain pH value. The reaction was monitored
by an analytical RP C8 column (Kromasil C8, The Nest Group, Inc.,
Southborough, MA, USA) under UV214 nm wavelength. After it appeared
complete, the reaction was quenched by adding 0.05% TFA to adjust
the pH to 7.0. The final product was then purified by semipreparative
HPLC at a flow rate of 4 mL/min. The ligated peptide was lyophilized
and characterized by an Agilent LC-MSD TOF. The preparative conditions
of MAPs are listed in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S10</ext-link>.</p><p>Detailed conditions of each MAPs peptide are summarized: MAP-S<sub>471–597</sub> (0.3 mg of BrK<sub>2</sub>KA reacted with 5.0
mg of S<sub>471–503</sub> peptide), MeCN/H<sub>2</sub>O/DMSO
(40:50:10 v/v/v) as mixture solvent, Na<sub>2</sub>CO<sub>3</sub> adjusting
pH 8.0, sonication assisting reaction, 30% yield, HPLC preparation
by a gradient of 0–25% MeCN against water for 5 min, 25–40%
MeCN for 50 min; MAP-S<sub>604–625</sub> (0.4 mg BrK<sub>2</sub>KA reacted with 5.1 mg of S<sub>604–625</sub> peptide), MeCN/H<sub>2</sub>O (25:75, v/v), Na<sub>2</sub>CO<sub>3</sub> adjusting pH
7.8, 40% yield, HPLC preparation by a gradient of 0–25% MeCN
against water for 5 min, 25–40% MeCN for 50 min; MAP-S<sub>597–625</sub> (0.35 mg of BrK<sub>2</sub>KA reacted with 5.0
mg of S<sub>597–625</sub> peptide), MeCN/H<sub>2</sub>O (50:50,
v/v), Na<sub>2</sub>CO<sub>3</sub> adjusting pH 8.5, 50% yield, HPLC
preparation by a gradient of 0–25% MeCN against water for 5
min, 25–40% MeCN for 50 min; MAP-S<sub>1164–1191</sub>(0.35 mg of BrK<sub>2</sub>KA reacted with 5.0 mg of S<sub>1164–1191</sub> peptide), MeCN/H<sub>2</sub>O (50:50, v/v), Na<sub>2</sub>CO<sub>3</sub> adjusting pH 8.5, 42% yield, HPLC preparation by a gradient
of 0–35% MeCN against water for 5 min, 35–40% MeCN for
50 min. Water and MeCN containing 0.1% TFA used for HPLC preparation
were preprepared. The column and flow rate were a Vydac 208TP510 and
4 mL/min, respectively.</p></sec><sec id="sec4.3"><title>PEPscan (Peptide Screening) for Linear B-Cell
Epitopes of SARS-CoV Recognized by Human Sera from SARS-CoV Recovered
Patients</title><p>The studies of human sera to evaluate their levels
of anti-SARS-CoV IgG that recognized the peptides were approved with
an Expedited Review of Research Plan by the Institutional Review Board
of Peking Union Medical College Hospital, Chinese Academy Medical
Sciences & Peking Union Medical College (CAMS & PUMC). More
than 500 antisera of SARS patients and 27 sera of healthy volunteers
were involved in this study, which was conducted according to the
guidelines for treatment of human subjects of the National Institutes
of Health, USA. Written informed consent was obtained from all study
participants.</p><p>The PEPscan was carried out as described by Hu
et al.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> without major modification. The
optical density was read at 450 and 630 nm in an optical density reader
(Labsystem, Franklin, MA, USA). The cutoff value was determined as
twice the average value of the negative controls.</p></sec><sec id="sec4.4"><title>Binding of
Mouse Monoclonal Antibodies to the SARS-CoV Virion</title><p>The SARS-CoV
PUMC01 strain<sup><xref ref-type="bibr" rid="ref46">46</xref></sup> was diluted with coating
buffer to 4 × 10<sup>–5</sup> TCID<sub>50</sub>/mL (bicarbonate/carbonate
coating buffer, 0.05 M, pH 9.6). The virus was then coated on 96-well
plates with 100 μL (4 × 10<sup>–4</sup> of TCID<sub>50</sub>) of virus per well for 12 h at 4 °C. The coating buffer
was removed from the wells, and 100 μL of blocking buffer was
added per well and incubated for 12 h at 4 °C (2% BSA in PBS,
pH 7.4; GIBCO, Carlsbad, CA, USA). The plates were washed twice with
PBS (pH 7.2). The mouse monoclonal antibodies against SARS-CoV and
control mAb against HIV-P27 were diluted to 0.001 μg/μL
in the blocking buffer, added to the wells separately, and incubated
for 1 h at 37 °C. The plates were washed twice with PBS and the
secondary antibody (HRP-conjugated goat anti-mouse IgG, (GeneTex,
Irvine, CA, USA) diluted 1:1000 in blocking buffer) was added to each
well and incubated for 1 h at 37 °C. The secondary antibody was
removed, the plate was washed three times with PBS, and then 100 μL
of TMB substrate solution (Promega, Madison, WI, USA) was added to
each well. After incubation for 30 min at room temperature, the absorbance
of the test wells was read at 450 nm (<italic>A</italic><sub>450</sub>).</p></sec><sec id="sec4.5"><title>Analysis of Neutralization Using a Neutral Red Staining (NRS)-Based
Assay</title><p>MAbs were serially diluted in DMEM culture medium.
Human polyclonal antisera from convalescent SARS patients were diluted
to 1:500 (v/v). The SARS-CoV PUMC01 strain was diluted with DMEM to
4 × 10<sup>–4</sup> TCID<sub>50</sub>. The NRS neutralization
assay was performed in 96-well flat-bottom plates. The setup is similar
to that of the cytopathic effect (CPE)-based neutralization assay.
After incubation of 50 μL (4 × 10<sup>–4</sup> TCID<sub>50</sub>) of virus with 50 μL of antibodies or antisera in
a total of 100 μL of DMEM medium per well for 1 h at 37 °C,
Vero E6 cells (2 × 10<sup>5</sup> cells in 100 μL, from
Cell Culture Center of PUMC) were added to each well. On day 5 of
infection, when >70% of the cells showed CPE in the viral control
wells (with DMEM instead of antibody or antisera), the culture medium
was removed from the test wells and 100 μL of 0.15% neutral
red (Sigma, St. Louis, MO, USA) in DMEM was added to each well. After
incubation for 1 h at 37 °C, the neutral red medium was removed,
the plates were washed twice with PBS (pH 7.2), and then 100 μL
of acidified alcohol (1% acetic acid in 50% ethanol) was added to
each well. After incubation for 30 min at room temperature, the absorbance
of neutral red stained plates was read at 450 nm (<italic>A</italic><sub>450</sub>).</p><p>Percent neutralization was determined by the
following formula: % neutralization = (<italic>A</italic><sub>450</sub> of antibody test wells – <italic>A</italic><sub>450</sub> of viral control wells)/(<italic>A</italic><sub>450</sub> of cell
control wells – <italic>A</italic><sub>450</sub> of viral control
wells) × 100.</p></sec><sec id="sec4.6"><title>Generation, Purification, and Measurement
of the Affinity of Antipeptide Monoclonal Antibodies</title><p>Ganp-transgenic
mice were originally established in a C57BL/6 strain as described
previously.<sup><xref ref-type="bibr" rid="ref47">47</xref></sup> The mice were backcrossed
with BALB/c mice for at least eight generations and used for immunization
to establish mAbs. All mice were maintained in specific pathogen-free
conditions by the Center for Animal Resources and Development (CARD),
Kumamoto University.</p><p>As immunogens, four peptides of S<sub>471–503</sub>, S<sub>604–625</sub>, S<sub>597–625</sub>, and S<sub>1164–1191</sub> were synthesized and conjugated with keyhole
limpet hemocyanin (KLH) by Operon Biotechnologies (Tokyo, Japan).
One hundred micrograms of peptide–KLH emulsified in complete
Freund’s adjuvant (CFA) was injected subcutaneously as a primary
immunization and boosted after 2 weeks with incomplete Freund’s
adjuvant (IFA). Sera of immunized mice were measured by ELISA using
plates coated with free peptides. The mice with highest serum Ab titers
were further immunized, and 4 days later the spleen cells were obtained
for polyethylene glycol-mediated cell fusion with mouse myeloma cell
line X63 using standard procedures.<sup><xref ref-type="bibr" rid="ref48">48</xref></sup> The
fused cells were selected with HAT medium in microculture plates at
a concentration of 2 × 10<sup>4</sup> cells/well in the presence
of IL-6 (5 units/mL).</p><p>The mAbs binding to the immunizing peptide
Ags were detected with HRP-conjugated goat anti-mouse IgG Ab (Zymed,
South San Francisco, CA, USA) with the substrate OPD (Wako Chemicals,
Osaka, Japan). The positive signals (<italic>A</italic><sub>490</sub> > 1.0) were selected, and hybridomas were cloned by a limiting
dilution method, adapted to serum-free media, and purified by protein
G-Sepharose affinity chromatography (GE Healthcare, Buckinghamshire,
UK). The purity of the antibodies was examined by SDS-PAGE and protein
staining with Coomassie Brilliant Blue. The heavy and light chain
isotypes were determined using an isotyping kit (BD Biosciences, Franklin
Lakes, NJ, USA).</p><p>The affinity of the mAbs for peptides was determined
by the BIAcore assay.<sup><xref ref-type="bibr" rid="ref49">49</xref></sup> The on- and off-rate
constants (<italic>k</italic><sub>on</sub> and <italic>k</italic><sub>off</sub>) for binding of the mAbs to S<sub>471–503</sub>,
S<sub>604–625</sub>, S<sub>597–625</sub>, and S<sub>1164–1191</sub> peptides (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref>) were determined using the BIAcore system
(BIAcore International AB, Uppsala, Sweden). The carboxyl-methylated
dextran surface of the sensor chip was activated with <italic>N</italic>-ethyl-<italic>N</italic>′-(3-dietylaminopropyl)carbodiimide
and <italic>N</italic>-hydroxysuccinimide (EDC–EHS).<sup><xref ref-type="bibr" rid="ref50">50</xref></sup> Each peptide was immobilized through the free
thiol group of a cysteine residue that was deliberately placed at
the N-terminus, by injection of 35 μL of a 20 μg/mL solution
in 10 mM 2-(<italic>N</italic>-morpholino)ethanesulfonic acid buffer
(pH 6) to the EDC–NHS-activated surface that had been reacted
with 2-(2-pyridinyldithio)ethaneamine. The excess disulfide groups
were deactivated by the addition of cysteine. The mAbs were diluted
in 10 mM Hepes (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.05% (v/v)
BIAcore surfactant P20 and injected over the immobilized antigen at
a flow rate of 5 μL/min. The association was monitored by the
increase in the refractive index of the sensor chip surface per unit
time. The dissociation of the mAbs was monitored after the end of
the association phase with a flow rate of 50 μL/min. Kinetic
rate constants were calculated from the collected data using the Pharmacia
Kinetics Evaluation software.<sup><xref ref-type="bibr" rid="ref51">51</xref></sup> The <italic>k</italic><sub>on</sub> was determined by measuring the rate of binding
to the antigen at different protein concentrations.</p></sec><sec id="sec4.7"><title>Macaque Experiments</title><sec id="sec4.7.1"><title>Animals</title><p>Rhesus monkeys (3–4 years old, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Tables S4 and S6</ext-link>, from the Institute of Laboratory Animal Science)
were examined according to the national microbiologic and parasite
SPF standard and were determined free of anti-SARS antibody. The SPF
monkeys were housed in an AAALAC-accredited facility before infection
with SARS-CoV. The monkeys were then housed in a biosafety level 3
(BSL-3) facility for infection experiments. All procedures were approved
by the Animal Use and Care Committee of the Institute of Laboratory
Animal Science, Peking Union Medical College (Animal Welfare Assurance
No. A5655-01, IACUC approval date September 24, 2007).</p></sec><sec id="sec4.8"><title>Challenges
with SARS-CoV</title><p>Monkey challenges were performed in a BSL-3
facility.<sup><xref ref-type="bibr" rid="ref37">37</xref></sup> Briefly, the animals were anesthetized
with ketamine (0.5 mL/kg, im). The TCID<sub>50</sub> 10<sup>–6</sup> (4.0 mL/monkey) of the stock PUMC01 strain virus was sprayed into
the nasal cavity of the animals.</p></sec><sec id="sec4.9"><title>Pathologic Examination</title><p>The lung tissues were sampled from the euthanized animals. Organs
were examined grossly, and photographs were taken to record the lung
damage.</p><p>Samples of 5–10 g of lung tissues were collected
from each lung and frozen at −80 °C. The remaining tissues
were fixed in 10% formaldehyde for 1 week in the BSL-3 laboratory.
The tissues were then processed in the regular pathology laboratory
by dehydration, embedding, and sectioning.</p><p>For hematoxylin and
eosin (H&E) staining, the sections were sequentially treated with
xylene I, 10 min; xylene II, 10 min; 100% alcohol, 5 min; 95% alcohol,
5 min; 90% alcohol, 5 min; 80% alcohol, 5 min; 70% alcohol, 5 min;
and PBS, 5 min. The sections were then stained in hematoxylin for
5 min; washed with tap water and double-distilled water for 5 s, with
PBS for 5 min, with 95% alcohol for 30 s, with eosin for 30 s, with
95% alcohol for 5 s, with 100% alcohol I for 5 s, with 100% alcohol
II for 5 s, with xylene I for 5 min, with xylene II for 5 min, and
with xylene III for 5 min; and mounted with mounting buffer (ZhongShan
Inc., Guangzhou, China).</p></sec><sec id="sec4.10"><title>Immunohistochemical Analysis of the Infected
Lung Tissues</title><p>A mixture of the mouse monoclonal antibodies
mAb-4E5 (0.01 μg/mL), mAb-11B1 (0.01 μg/mL), and mAb-9A6
(0.01 μg/mL) was used as primary antibody for the immunohistochemical
analysis of the infected lung tissues. Briefly, tissue sections were
treated with the primary antibody at 4 °C overnight, washed three
times with PBS, incubated with HRP–goat-anti-mouse IgG (1:1000,
ZhongShan Inc., PRC) at 37 °C for 30 min, and then washed three
times with PBS. The DAB substrate (1:50) was added and incubated at
37 °C for 30 min and then stained with hematoxylin for 3 min.</p></sec><sec id="sec4.11"><title>RT-PCR for Analysis of the SARS-CoV Burden</title><p>The tissues from
cranial and caudal lobes of the left lung and cranial, middle, and
caudal lobes of the right lung from each animal were sampled and pooled
together, and the total RNA was isolated from the lung tissues using
Trizol (Invitrogen, USA). One hundred milligrams of lung tissue was
mixed with 1 mL of Trizol extraction buffer and homogenized for 60
s (Homogenizer, IKA, Germany). The RNA wan then extracted with chloroform
and precipitated with isopropanol. RNA was dissolved in 50 μL
of DEPC water, and the RNA quality was verified by electrophoresis
on agarose gel.</p><p>For the reverse transcription, 5 μg of
total RNA was reverse transcribed with a high-capacity cDNA kit (RecertAid
First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA)
with random hexamer primers. The reaction was performed at 42 °C
for 60 min and terminated at 70 °C for 10 min.</p><p>Two microliters
of reverse transcription mixture was used as template. The primers
for SARS-CoV were 5′-ATGAATTACCAAGTCAATGGTTAC-3′
and 5′-CATAACCAGTCGGTACAGCTAC-3′.
The PCR was normalized against monkey glyceraldehyde phosphate dehydrogenase
(GAPDH) (NM002046). The GAPDH primers were 5′-TCAACAGCGACACCCACTC-3′
and 5′-CTTCCTCTTGTGCTCTTGCTG-3′
(product size = 201 bp). The PCR was performed with a PCR kit (<italic>Taq</italic> DNA polymerase, Fermentas). The PCR products were detected
by 1.6% agarose gel electrophoresis.</p><p>Reaction conditions were
as follows: 10× <italic>Taq</italic> buffer (2 μL), 2 mM
dNTP (2 μL), forward primer (20 μM, 0.4 μL), reverse
primer (20 μM, 0.4 μL), <italic>Taq</italic> DNA polymerase
(0.8 μL), 25 mM MgCl<sub>2</sub> (2 μL), template DNA
(cDNA, 2 μL), H<sub>2</sub>O (9.4 μL); step 1, 94 °C
for 3 min; step 2, 94 °C for 30 s, 55 °C for 30 s, 72 °C
for 30 s, for 26 cycles, then 72 °C for 10 min.</p></sec><sec id="sec4.12"><title>qRT-PCR for
Analysis of the SARS-CoV Burden</title><p>The SARS-CoV fragment was
prepared by RT-PCR. A 240 bp fragment of SARS-CoV was amplified and
separated by 1.6% agarose electrophoresis. The DNA fragment was recovered
from the gel with a gel extraction kit (MinElute Gel Extraction Kit,
QIAGEN Stanford, Valencia, CA, USA). The recovered DNA was measured
and adjusted to concentrations of 1 × 10<sup>6</sup>, 1 ×
10<sup>5</sup>, 1 × 10<sup>4</sup>, 1 × 10<sup>3</sup>,
1 × 10<sup>2</sup>, and 1 × 10<sup>1</sup> copies/μL,
which were used to generate a standard curve. This known copy number
standard curve was used to calculate the copy number of SARS-CoV mRNA
in the infected lung tissues.</p><p>RT-PCR for the infected samples
was performed under the same conditions used for standard curves with
the Roche Light Cycler following the instructions for the SYBR Green
Realtime PCR Master Mix (Toyobo, Osaka, Japan).</p><p>The copy number
of the virus was detected as follows. Briefly, 2 μL of the reverse
transcription mixture from step 1 was used as template. The primers
for SARS-CoV were 5′-ATGAATTACCAAGTCAATGGTTAC-3′
and 5′-CATAACCAGTCGGTACAGCTAC-3′.
The reaction was continued for 40 cycles under the conditions: 2 ×
QuantiTect SYBR Green PCR (10 μL), gorward primer (10 μM,
1 μL), teverse primer (10 μM, 1 μL), RNase-free
water (3 μL), yemplate DNA (cDNA, 5 μL), total volume
(20 μL); step 1, 94 °C for 3 min; step 2, 94 °C for
15 s, 55 °C for 30 s, 72 °C for 20 s, for 40 cycles and
then move to the melt curve procedure.</p></sec><sec id="sec4.13"><title>Immunization of Monkeys</title><p>The peptide and/or peptide mixtures were dissolved as 480 μg/mL
of each peptide in 0.9% NaCl and then formulated with an equal volume
of Freund’s Complete Adjuvant (FCA) (Sigma, St. Louis, MO,
USA) according to the manufacturer’s instructions. The injected
dose of vaccine was 0.5 mL/4 kg/monkey (120 μg/4 kg/monkey)
given through intramuscular (im) injections at four sites on the arms
and legs.</p><p>Rhesus monkeys were boosted at 2, 4, and 6 weeks after
the first injection with the same dose of peptides and volume of vaccine
as for the first injection, but formulated in an equal volume of Freund’s
incomplete adjuvant (IFA) (Sigma).</p><p>Venous blood samples (2 mL/each)
were collected every 7 days from each monkey. The sera were isolated
by centrifugation at 2000 rpm and immediately stored at −80
°C.</p><p>Antipeptidic IgG was detected by ELISA. Free peptides
were diluted in carbonate buffer (pH 9.6) to a final concentration
of 10 μg/mL. One hundred microliters of the peptide solution
was added to 96-well polystyrene high BIND microplates (Corning Life
Science, Santa Clara, CA, USA) for coating overnight at 4 °C.
The wells of plates were then washed three times with PBS-Tween buffer
(10 mM phosphate buffer, 150 mM NaCl, 0.05% Tween 20, pH 7.2) and
subsequently blocked with 3% BSA in PBS-Tween buffer for an additional
1 h at 37 °C. After three thorough washings of the wells with
PBS-Tween buffer, the antigen-coated microplates were incubated with
diluted monkey sera at room temperature for 1 h. After three washings
with PBS-Tween buffer, bound antibodies were detected with HRP-conjugated
goat anti-monkey IgG (1:2000 v/v, Gene Tex). After three washings
with PBS-Tween, a TMB substrate solution (Promega, Madison, WI, USA)
was added and the color developed for 20 min before the reaction was
stopped with 100 μL of 0.2 M H<sub>2</sub>SO<sub>4</sub>. The
absorbance at 450 nm was read using a microtiter plate reader.</p></sec><sec id="sec4.14"><title>Macaques
Treated by MAb43-3-14</title><p>Eighteen rhesus monkeys in six groups
(<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">Table S6</ext-link>, 3–4 years old) were examined
according to the national microbiologic and parasite SPF standard
and were verified free of anti-SARS antibody. mAb43-3-14 was adjusted
to concentrations of 0.4 and 3.6 mg/mL with 0.9% NaCl, respectively.
A dose of 0.2 or 1.8 mg/kg of the antibody given by intravenous (iv)
injection was used to treat the different groups. The same volume
of 0.9% NaCl was used for the control group. The procedures were approved
by the Animal Use and Care Committee of the Institute of Laboratory
Animal Science, Peking Union Medical College (Animal Welfare Assurance
No. A5655-01, IACUC approval date September 24, 2007).</p></sec></sec><sec id="sec4.15"><title>Statistical
Analysis</title><p>All data were analyzed using Prism software (GraphPad
software). For neutralization assays and ELISA, mean and standard
deviation were used to determine significance. For qRT-PCR, geometric
mean and standard deviation were used to determine virus burden.</p></sec></sec></body><back><notes id="notes-1" notes-type="si"><title>Supporting Information Available</title><p>The
Supporting Information is available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link> at
DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acsinfecdis.6b00006">10.1021/acsinfecdis.6b00006</ext-link>.<list id="silist" list-type="simple"><list-item><p>Neutralization
and enhancement of SARS-CoV infection of Vero E6 cells in the presence
of human antisera of convalescent SARS patients, design and synthesis
of new peptides, generated antipeptide mAbs, monkeys for peptide vaccine
immunization against SARS-CoV, pathologic classification of the severity
of the lung damage in SARS-CoV-infected rhesus macaques, conditions
for preparation of multiple antigen peptides (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsinfecdis.6b00006/suppl_file/id6b00006_si_001.pdf">PDF</ext-link>)</p></list-item></list></p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="id6b00006_si_001.pdf"><caption><p>id6b00006_si_001.pdf</p></caption></media></supplementary-material></sec><notes notes-type="" id="notes-2"><title>Author Contributions</title><p><sup>∥</sup> W.Q.D.,
Z.L.F., and K.K. equally contributed to this work.</p></notes><notes notes-type="COI-statement" id="notes-3"><p>The authors declare
no competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>We are grateful
to Professor Carl F. Nathan and Dr. Michael S. Diamond for their kind
discussion and manuscript preparation. We thank Dr. Baoxing Fan for
his serological evaluation of the large number of SARS-CoV convalescent
antisera. The project described in this paper was supported by the
National Institute of Allergy and Infectious Diseases (U01AI061092).
The study was also partially supported for preparation of the monoclonal
antibodies by contract research for MEXT for Emerging and Reemerging
Infectious Diseases and Promotion of Fundamental Studies in Health
Sciences (04-2) of the NIBIO and National Natural Science Foundation
of China (No. 90713045). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation
of the manuscript.</p></ack><ref-list><title>References</title><ref id="ref1"><mixed-citation publication-type="journal" id="cit1"><name><surname>Hilgenfeld</surname><given-names>R.</given-names></name>; <name><surname>Peiris</surname><given-names>M.</given-names></name> (<year>2013</year>) <article-title>From SARS
to MERS: 10 years of research on highly pathogenic human coronavirus</article-title>. <source>Antiviral Res.</source><volume>100</volume> (<issue>1</issue>), <fpage>286</fpage>–<lpage>295</lpage>. <pub-id pub-id-type="doi">10.1016/j.antiviral.2013.08.015</pub-id>.<pub-id pub-id-type="pmid">24012996</pub-id></mixed-citation></ref><ref id="ref2"><mixed-citation publication-type="journal" id="cit2"><name><surname>Drosten</surname><given-names>C.</given-names></name>; <name><surname>Günther</surname><given-names>S.</given-names></name>; <name><surname>Preiser</surname><given-names>W.</given-names></name>; <name><surname>van der Werf</surname><given-names>S.</given-names></name>; <name><surname>Brodt</surname><given-names>H. R.</given-names></name>; et al. (<year>2003</year>) <article-title>Identification
of a novel coronavirus in patients with severe acute respiratory syndrome</article-title>. <source>N. Engl. J. Med.</source><volume>348</volume>, <fpage>1967</fpage>–<lpage>1976</lpage>. <pub-id pub-id-type="doi">10.1056/NEJMoa030747</pub-id>.<pub-id pub-id-type="pmid">12690091</pub-id></mixed-citation></ref><ref id="ref3"><mixed-citation publication-type="journal" id="cit3"><name><surname>Li</surname><given-names>W.</given-names></name>; <name><surname>Shi</surname><given-names>Z.</given-names></name>; <name><surname>Yu</surname><given-names>M.</given-names></name>; <name><surname>Ren</surname><given-names>W.</given-names></name>; <name><surname>Smith</surname><given-names>C.</given-names></name>; et al. (<year>2005</year>) <article-title>Bats are
natural reservoirs of SARS-like coronaviruses</article-title>. <source>Science</source><volume>310</volume>, <fpage>676</fpage>–<lpage>679</lpage>. <pub-id pub-id-type="doi">10.1126/science.1118391</pub-id>.<pub-id pub-id-type="pmid">16195424</pub-id></mixed-citation></ref><ref id="ref4"><mixed-citation publication-type="journal" id="cit4"><name><surname>Guan</surname><given-names>Y.</given-names></name>; <name><surname>Zheng</surname><given-names>B. J.</given-names></name>; <name><surname>He</surname><given-names>Y. Q.</given-names></name>; <name><surname>Liu</surname><given-names>X. L.</given-names></name>; <name><surname>Zhuang</surname><given-names>Z. X.</given-names></name>; <name><surname>Cheung</surname><given-names>C. L.</given-names></name>; et al. (<year>2003</year>) <article-title>Isolation and characterization of viruses related to the SARS coronavirus
from animals in southern China</article-title>. <source>Science</source><volume>302</volume>, <fpage>276</fpage>–<lpage>278</lpage>. <pub-id pub-id-type="doi">10.1126/science.1087139</pub-id>.<pub-id pub-id-type="pmid">12958366</pub-id></mixed-citation></ref><ref id="ref5"><mixed-citation publication-type="journal" id="cit5"><name><surname>Marra</surname><given-names>M. A.</given-names></name>; <name><surname>Jones</surname><given-names>S. J.</given-names></name>; <name><surname>Astell</surname><given-names>C. R.</given-names></name>; <name><surname>Holt</surname><given-names>R. A.</given-names></name>; <name><surname>Brooks-Wilson</surname><given-names>A.</given-names></name>; et al. (<year>2003</year>) <article-title>The genome sequence
of the SARS-associated coronavirus</article-title>. <source>Science</source><volume>300</volume>, <fpage>1399</fpage>–<lpage>1404</lpage>. <pub-id pub-id-type="doi">10.1126/science.1085953</pub-id>.<pub-id pub-id-type="pmid">12730501</pub-id></mixed-citation></ref><ref id="ref6"><mixed-citation publication-type="journal" id="cit6"><name><surname>Rota</surname><given-names>P. A.</given-names></name>; <name><surname>Oberste</surname><given-names>M. S.</given-names></name>; <name><surname>Monroe</surname><given-names>S. S.</given-names></name>; <name><surname>Nix</surname><given-names>W. A.</given-names></name>; <name><surname>Campagnoli</surname><given-names>R.</given-names></name>; et al. (<year>2003</year>) <article-title>Characterization of
a novel coronavirus associated with severe acute respiratory syndrome</article-title>. <source>Science</source><volume>300</volume>, <fpage>1394</fpage>–<lpage>1399</lpage>. <pub-id pub-id-type="doi">10.1126/science.1085952</pub-id>.<pub-id pub-id-type="pmid">12730500</pub-id></mixed-citation></ref><ref id="ref7"><note id="cit7"><p>The Chinese SARS molecular epidemiology consortium</p></note></ref><ref id="ref8"><mixed-citation publication-type="journal" id="cit8"><name><surname>Li</surname><given-names>W.</given-names></name>; <name><surname>Moore</surname><given-names>M. J.</given-names></name>; <name><surname>Vasilieva</surname><given-names>N.</given-names></name>; <name><surname>Sui</surname><given-names>J.</given-names></name>; <name><surname>Wong</surname><given-names>S. K.</given-names></name>; et al. (<year>2003</year>) <article-title>Angiotensin-converting enzyme 2 is a functional
receptor for the SARS coronavirus</article-title>. <source>Nature</source><volume>426</volume>, <fpage>450</fpage>–<lpage>454</lpage>. <pub-id pub-id-type="doi">10.1038/nature02145</pub-id>.<pub-id pub-id-type="pmid">14647384</pub-id></mixed-citation></ref><ref id="ref9"><mixed-citation publication-type="journal" id="cit9"><name><surname>Jeffers</surname><given-names>S. A.</given-names></name>; <name><surname>Tusell</surname><given-names>S. M.</given-names></name>; <name><surname>Gillim-Ross</surname><given-names>L.</given-names></name>; <name><surname>Hemmila</surname><given-names>E. M.</given-names></name>; <name><surname>Achenbach</surname><given-names>J. E.</given-names></name>; et al. (<year>2004</year>) <article-title>CD209L (L-SIGN) is a
receptor for severe acute respiratory syndrome coronavirus</article-title>. <source>Proc. Natl. Acad. Sci. U.S.A.</source><volume>101</volume>, <fpage>15748</fpage>–<lpage>15753</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.0403812101</pub-id>.<pub-id pub-id-type="pmid">15496474</pub-id></mixed-citation></ref><ref id="ref10"><mixed-citation publication-type="journal" id="cit10"><name><surname>Wang</surname><given-names>B.</given-names></name>; <name><surname>Chen</surname><given-names>H.</given-names></name>; <name><surname>Jiang</surname><given-names>X.</given-names></name>; <name><surname>Zhang</surname><given-names>M.</given-names></name>; <name><surname>Wan</surname><given-names>T.</given-names></name>; et al. (<year>2004</year>) <article-title>Identification of an
HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike
protein</article-title>. <source>Blood</source><volume>104</volume>, <fpage>200</fpage>–<lpage>206</lpage>. <pub-id pub-id-type="doi">10.1182/blood-2003-11-4072</pub-id>.<pub-id pub-id-type="pmid">15016646</pub-id></mixed-citation></ref><ref id="ref11"><mixed-citation publication-type="journal" id="cit11"><name><surname>Wang</surname><given-names>Y. D.</given-names></name>; <name><surname>Sin</surname><given-names>W. Y.</given-names></name>; <name><surname>Xu</surname><given-names>G. B.</given-names></name>; <name><surname>Yang</surname><given-names>H. H.</given-names></name>; <name><surname>Wong</surname><given-names>T. Y.</given-names></name>; et al. (<year>2004</year>) <article-title>T-cell epitopes in severe
acute respiratory syndrome (SARS) coronavirus spike protein elicit
a specific T-cell immune response in patients who recover from SARS</article-title>. <source>J. Virol.</source><volume>78</volume>, <fpage>5612</fpage>–<lpage>5618</lpage>. <pub-id pub-id-type="doi">10.1128/JVI.78.11.5612-5618.2004</pub-id>.<pub-id pub-id-type="pmid">15140958</pub-id></mixed-citation></ref><ref id="ref12"><mixed-citation publication-type="journal" id="cit12"><name><surname>Roberts</surname><given-names>A.</given-names></name>; <name><surname>Wood</surname><given-names>J.</given-names></name>; <name><surname>Subbarao</surname><given-names>K.</given-names></name>; <name><surname>Ferguson</surname><given-names>M.</given-names></name>; <name><surname>Wood</surname><given-names>D.</given-names></name>; et al. (<year>2006</year>) <article-title>Animal models and antibody
assays for evaluating candidate SARS vaccines: summary of a technical
meeting 25–26 August 2005, London, UK</article-title>. <source>Vaccine</source><volume>24</volume>, <fpage>7056</fpage>–<lpage>7065</lpage>. <pub-id pub-id-type="doi">10.1016/j.vaccine.2006.07.009</pub-id>.<pub-id pub-id-type="pmid">16930781</pub-id></mixed-citation></ref><ref id="ref13"><mixed-citation publication-type="journal" id="cit13"><name><surname>Gillim-Ross</surname><given-names>L.</given-names></name>; <name><surname>Subbarao</surname><given-names>K.</given-names></name> (<year>2006</year>) <article-title>Emerging respiratory viruses: challenges and vaccine
strategies</article-title>. <source>Clin. Microbiol. Rev.</source><volume>19</volume>, <fpage>614</fpage>–<lpage>636</lpage>. <pub-id pub-id-type="doi">10.1128/CMR.00005-06</pub-id>.<pub-id pub-id-type="pmid">17041137</pub-id></mixed-citation></ref><ref id="ref14"><mixed-citation publication-type="journal" id="cit14"><name><surname>Graham</surname><given-names>R. L.</given-names></name>; <name><surname>Donaldson</surname><given-names>E. F.</given-names></name>; <name><surname>Baric</surname><given-names>R. S.</given-names></name> (<year>2013</year>) <article-title>A decade after SARS: strategies for controlling emerging
coronaviruses</article-title>. <source>Nat. Rev. Microbiol.</source><volume>11</volume> (<issue>12</issue>), <fpage>836</fpage>–<lpage>848</lpage>. <pub-id pub-id-type="doi">10.1038/nrmicro3143</pub-id>.<pub-id pub-id-type="pmid">24217413</pub-id></mixed-citation></ref><ref id="ref15"><mixed-citation publication-type="journal" id="cit15"><name><surname>Yang</surname><given-names>Z. Y.</given-names></name>; <name><surname>Werner</surname><given-names>H. C.</given-names></name>; <name><surname>Kong</surname><given-names>W. P.</given-names></name>; <name><surname>Leung</surname><given-names>K.</given-names></name>; <name><surname>Traggiai</surname><given-names>E.</given-names></name>; et al. (<year>2005</year>) <article-title>Evasion of antibody neutralization in emerging severe acute respiratory
syndrome coronaviruses</article-title>. <source>Proc. Natl. Acad. Sci.
U.S.A.</source><volume>102</volume>, <fpage>797</fpage>–<lpage>801</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.0409065102</pub-id>.<pub-id pub-id-type="pmid">15642942</pub-id></mixed-citation></ref><ref id="ref16"><mixed-citation publication-type="journal" id="cit16"><name><surname>Dandekar</surname><given-names>A. A.</given-names></name>; <name><surname>Perlman</surname><given-names>S.</given-names></name> (<year>2005</year>) <article-title>Immunopathogenesis of coronavirus infections: implications
for SARS</article-title>. <source>Nat. Rev. Immunol.</source><volume>5</volume>, <fpage>917</fpage>–<lpage>927</lpage>. <pub-id pub-id-type="doi">10.1038/nri1732</pub-id>.<pub-id pub-id-type="pmid">16322745</pub-id></mixed-citation></ref><ref id="ref17"><mixed-citation publication-type="journal" id="cit17"><name><surname>Subbarao</surname><given-names>K.</given-names></name>; <name><surname>Roberts</surname><given-names>A.</given-names></name> (<year>2006</year>) <article-title>Is there an ideal animal
model for SARS?</article-title>. <source>Trends Microbiol.</source><volume>14</volume>, <fpage>299</fpage>–<lpage>303</lpage>. <pub-id pub-id-type="doi">10.1016/j.tim.2006.05.007</pub-id>.<pub-id pub-id-type="pmid">16759866</pub-id></mixed-citation></ref><ref id="ref18"><mixed-citation publication-type="journal" id="cit18"><name><surname>Taylor</surname><given-names>D. R.</given-names></name> (<year>2006</year>) <article-title>Obstacles and advances in SARS vaccine
development</article-title>. <source>Vaccine</source><volume>24</volume>, <fpage>863</fpage>–<lpage>871</lpage>. <pub-id pub-id-type="doi">10.1016/j.vaccine.2005.08.102</pub-id>.<pub-id pub-id-type="pmid">16191455</pub-id></mixed-citation></ref><ref id="ref19"><mixed-citation publication-type="journal" id="cit19"><name><surname>Takada</surname><given-names>A.</given-names></name>; <name><surname>Kawaoka</surname><given-names>Y.</given-names></name> (<year>2003</year>) <article-title>Antibody-dependent
enhancement of viral infection: molecular mechanisms and in vivo implications</article-title>. <source>Rev. Med. Virol.</source><volume>13</volume>, <fpage>387</fpage>–<lpage>398</lpage>. <pub-id pub-id-type="doi">10.1002/rmv.405</pub-id>.<pub-id pub-id-type="pmid">14625886</pub-id></mixed-citation></ref><ref id="ref20"><mixed-citation publication-type="journal" id="cit20"><name><surname>Tirado</surname><given-names>S. M. C.</given-names></name>; <name><surname>Yoon</surname><given-names>K. J.</given-names></name> (<year>2003</year>) <article-title>Antibody-dependent enhancement of virus infection and
disease</article-title>. <source>Viral Immunol.</source><volume>16</volume>, <fpage>69</fpage>–<lpage>86</lpage>. <pub-id pub-id-type="doi">10.1089/088282403763635465</pub-id>.<pub-id pub-id-type="pmid">12725690</pub-id></mixed-citation></ref><ref id="ref21"><mixed-citation publication-type="journal" id="cit21"><name><surname>Gorlani</surname><given-names>A.</given-names></name>; <name><surname>Forthal</surname><given-names>D. N.</given-names></name> (<year>2013</year>) <article-title>Antibody-dependent
enhancement and the risk of HIV infection</article-title>. <source>Curr.
HIV Res.</source><volume>11</volume> (<issue>5</issue>), <fpage>421</fpage>–<lpage>426</lpage>. <pub-id pub-id-type="doi">10.2174/1570162X113116660062</pub-id>.<pub-id pub-id-type="pmid">24191936</pub-id></mixed-citation></ref><ref id="ref22"><mixed-citation publication-type="journal" id="cit22"><name><surname>Sullivan</surname><given-names>N.</given-names></name>; <name><surname>Sun</surname><given-names>Y.</given-names></name>; <name><surname>Binley</surname><given-names>J.</given-names></name>; <name><surname>Lee</surname><given-names>J.</given-names></name>; <name><surname>Barbas</surname><given-names>C. F.</given-names><suffix>3rd</suffix></name>; <name><surname>Parren</surname><given-names>P. W.</given-names></name>; <name><surname>Burton</surname><given-names>D. R.</given-names></name>; <name><surname>Sodroski</surname><given-names>J.</given-names></name> (<year>1998</year>) <article-title>Determinants of human immunodeficiency virus type 1
envelope glycoprotein activation by soluble CD4 and monoclonal antibodies</article-title>. <source>J. Virol.</source><volume>72</volume> (<issue>8</issue>), <fpage>6332</fpage>–<lpage>6338</lpage>.<pub-id pub-id-type="pmid">9658072</pub-id></mixed-citation></ref><ref id="ref23"><mixed-citation publication-type="journal" id="cit23"><name><surname>Guillon</surname><given-names>C.</given-names></name>; <name><surname>Schutten</surname><given-names>M.</given-names></name>; <name><surname>Boers</surname><given-names>P. H.</given-names></name>; <name><surname>Gruters</surname><given-names>R. A.</given-names></name>; <name><surname>Osterhaus</surname><given-names>A. D.</given-names></name> (<year>2002</year>) <article-title>Antibody-mediated
enhancement of human immunodeficiency virus type 1 infectivity is
determined by the structure of gp120 and depends on modulation of
the gp120-CCR5 interaction</article-title>. <source>J. Virol.</source><volume>76</volume> (<issue>6</issue>), <fpage>2827</fpage>–<lpage>2834</lpage>. <pub-id pub-id-type="doi">10.1128/JVI.76.6.2827-2834.2002</pub-id>.<pub-id pub-id-type="pmid">11861850</pub-id></mixed-citation></ref><ref id="ref24"><mixed-citation publication-type="journal" id="cit24"><name><surname>Gorlani</surname><given-names>A.</given-names></name>; <name><surname>Forthal</surname><given-names>D. N.</given-names></name> (<year>2013</year>) <article-title>Antibody-dependent enhancement and the risk of HIV
infection</article-title>. <source>Curr. HIV Res.</source><volume>11</volume> (<issue>5</issue>), <fpage>421</fpage>–<lpage>426</lpage>. <pub-id pub-id-type="doi">10.2174/1570162X113116660062</pub-id>.<pub-id pub-id-type="pmid">24191936</pub-id></mixed-citation></ref><ref id="ref25"><mixed-citation publication-type="journal" id="cit25"><name><surname>Halstead</surname><given-names>S. B.</given-names></name> (<year>2007</year>) <article-title>Dengue</article-title>. <source>Lancet</source><volume>370</volume>, <fpage>1644</fpage>–<lpage>1652</lpage>. <pub-id pub-id-type="doi">10.1016/S0140-6736(07)61687-0</pub-id>.<pub-id pub-id-type="pmid">17993365</pub-id></mixed-citation></ref><ref id="ref26"><mixed-citation publication-type="journal" id="cit26"><name><surname>Goncalvez</surname><given-names>A. P.</given-names></name>; <name><surname>Engle</surname><given-names>R. E.</given-names><suffix>St</suffix></name>; <name><surname>Claire</surname><given-names>M.</given-names></name>; <name><surname>Purcell</surname><given-names>R. H.</given-names></name>; <name><surname>Lai</surname><given-names>C. J.</given-names></name> (<year>2007</year>) <article-title>Monoclonal antibody-mediated
enhancement of dengue virus infection in vitro and in vivo and strategies
for prevention</article-title>. <source>Proc. Natl. Acad. Sci. U.S.A.</source><volume>104</volume>, <fpage>9422</fpage>–<lpage>9427</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.0703498104</pub-id>.<pub-id pub-id-type="pmid">17517625</pub-id></mixed-citation></ref><ref id="ref27"><mixed-citation publication-type="journal" id="cit27"><name><surname>Pierson</surname><given-names>T. C.</given-names></name>; <name><surname>Xu</surname><given-names>Q.</given-names></name>; <name><surname>Nelson</surname><given-names>S.</given-names></name>; <name><surname>Oliphant</surname><given-names>T.</given-names></name>; <name><surname>Grant</surname><given-names>E.</given-names></name>; et al. (<year>2007</year>) <article-title>The stoichiometry of
antibody-mediated neutralization and enhancement of West Nile virus
infection</article-title>. <source>Cell Host Microbe</source><volume>1</volume>, <fpage>135</fpage>–<lpage>145</lpage>. <pub-id pub-id-type="doi">10.1016/j.chom.2007.03.002</pub-id>.<pub-id pub-id-type="pmid">18005691</pub-id></mixed-citation></ref><ref id="ref28"><mixed-citation publication-type="journal" id="cit28"><name><surname>Taylor</surname><given-names>A.</given-names></name>; <name><surname>Foo</surname><given-names>S. S.</given-names></name>; <name><surname>Bruzzone</surname><given-names>R.</given-names></name>; <name><surname>Dinh</surname><given-names>L. V.</given-names></name>; <name><surname>King</surname><given-names>N. J.</given-names></name>; <name><surname>Mahalingam</surname><given-names>S.</given-names></name> (<year>2015</year>) <article-title>Fc receptors
in antibody-dependent enhancement of viral infections</article-title>. <source>Immunol Rev.</source><volume>268</volume> (<issue>1</issue>), <fpage>340</fpage>–<lpage>364</lpage>. <pub-id pub-id-type="doi">10.1111/imr.12367</pub-id>.<pub-id pub-id-type="pmid">26497532</pub-id></mixed-citation></ref><ref id="ref29"><mixed-citation publication-type="journal" id="cit29"><name><surname>Pierson</surname><given-names>T. C.</given-names></name>; <name><surname>Sánchez</surname><given-names>M. D.</given-names></name>; <name><surname>Puffer</surname><given-names>B. A.</given-names></name>; <name><surname>Ahmed</surname><given-names>A. A.</given-names></name>; <name><surname>Geiss</surname><given-names>B. J.</given-names></name>; <name><surname>Valentine</surname><given-names>L. E.</given-names></name>; <name><surname>Altamura</surname><given-names>L. A.</given-names></name>; <name><surname>Diamond</surname><given-names>M. S.</given-names></name>; <name><surname>Doms</surname><given-names>R. W.</given-names></name> (<year>2006</year>) <article-title>A rapid and quantitative
assay for measuring antibody-mediated neutralization of West Nile
virus infection</article-title>. <source>Virology</source><volume>346</volume> (<issue>1</issue>), <fpage>53</fpage>–<lpage>65</lpage>. <pub-id pub-id-type="doi">10.1016/j.virol.2005.10.030</pub-id>.<pub-id pub-id-type="pmid">16325883</pub-id></mixed-citation></ref><ref id="ref30"><mixed-citation publication-type="journal" id="cit30"><name><surname>Mehlhop</surname><given-names>E.</given-names></name>; <name><surname>Ansarah-Sobrinho</surname><given-names>C.</given-names></name>; <name><surname>Johnson</surname><given-names>S.</given-names></name>; <name><surname>Engle</surname><given-names>M.</given-names></name>; <name><surname>Fremont</surname><given-names>D. H.</given-names></name>; <name><surname>Pierson</surname><given-names>T. C.</given-names></name>; <name><surname>Diamond</surname><given-names>M. S.</given-names></name> (<year>2007</year>) <article-title>Complement protein C1q inhibits antibody-dependent
enhancement of flavivirus infection in an IgG subclass-specific manner</article-title>. <source>Cell Host Microbe</source><volume>2</volume> (<issue>6</issue>), <fpage>417</fpage>–<lpage>426</lpage>. <pub-id pub-id-type="doi">10.1016/j.chom.2007.09.015</pub-id>.<pub-id pub-id-type="pmid">18078693</pub-id></mixed-citation></ref><ref id="ref31"><mixed-citation publication-type="journal" id="cit31"><name><surname>Huang</surname><given-names>K. J.</given-names></name>; <name><surname>Yang</surname><given-names>Y. C.</given-names></name>; <name><surname>Lin</surname><given-names>Y. S.</given-names></name>; <name><surname>Huang</surname><given-names>J. H.</given-names></name>; <name><surname>Lin</surname><given-names>H. S.</given-names></name>; <name><surname>Yeh</surname><given-names>T. M.</given-names></name>; <name><surname>Chen</surname><given-names>S. H.</given-names></name>; <name><surname>Liu</surname><given-names>C. C.</given-names></name>; <name><surname>Lei</surname><given-names>H. Y.</given-names></name> (<year>2006</year>) <article-title>The dual-specific binding of dengue
virus and target cells for the antibody-dependent enhancement of dengue
virus infection</article-title>. <source>J. Immunol.</source><volume>176</volume>, <fpage>2825</fpage>–<lpage>2832</lpage>. <pub-id pub-id-type="doi">10.4049/jimmunol.176.5.2825</pub-id>.<pub-id pub-id-type="pmid">16493039</pub-id></mixed-citation></ref><ref id="ref32"><mixed-citation publication-type="journal" id="cit32"><name><surname>He</surname><given-names>Y. X.</given-names></name>; <name><surname>Zhou</surname><given-names>Y.</given-names></name>; <name><surname>Siddiqui</surname><given-names>P.</given-names></name>; <name><surname>Niu</surname><given-names>J.</given-names></name>; <name><surname>Jiang</surname><given-names>S. B.</given-names></name> (<year>2005</year>) <article-title>Identification of immunodominant epitopes on the membrane
protein of the severe acute respiratory syndrome-associated coronavirus</article-title>. <source>J. Clin Microbiol.</source><volume>43</volume> (<issue>8</issue>), <fpage>3718</fpage>–<lpage>3726</lpage>. <pub-id pub-id-type="doi">10.1128/JCM.43.8.3718-3726.2005</pub-id>.<pub-id pub-id-type="pmid">16081901</pub-id></mixed-citation></ref><ref id="ref33"><mixed-citation publication-type="journal" id="cit33"><name><surname>He</surname><given-names>Y. X.</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>Kou</surname><given-names>Z. H.</given-names></name>; <name><surname>Liu</surname><given-names>S. W.</given-names></name>; <name><surname>Jiang</surname><given-names>S. B.</given-names></name> (<year>2004</year>) <article-title>Mapping of Antigenic
Sites on the Nucleocapsid Protein of the Severe Acute Respiratory
Syndrome Coronavirus</article-title>. <source>J. Clin Microbiol.</source><volume>42</volume> (<issue>11</issue>), <fpage>5309</fpage>–<lpage>5314</lpage>. <pub-id pub-id-type="doi">10.1128/JCM.42.11.5309-5314.2004</pub-id>.<pub-id pub-id-type="pmid">15528730</pub-id></mixed-citation></ref><ref id="ref34"><mixed-citation publication-type="journal" id="cit34"><name><surname>Liang</surname><given-names>Y. F.</given-names></name>; <name><surname>Wan</surname><given-names>Y.</given-names></name>; <name><surname>Qiu</surname><given-names>Li. W.</given-names></name>; <name><surname>Zhou</surname><given-names>J.</given-names></name>; <name><surname>Ni</surname><given-names>B.</given-names></name>; <name><surname>Guo</surname><given-names>B.</given-names></name>; <name><surname>Zou</surname><given-names>Q.</given-names></name>; <name><surname>Zou</surname><given-names>L. Y.</given-names></name>; <name><surname>Zhou</surname><given-names>W.</given-names></name>; <name><surname>Jia</surname><given-names>Z. C.</given-names></name>; <name><surname>Che</surname><given-names>X. Y.</given-names></name>; <name><surname>Wu</surname><given-names>Y. Z.</given-names></name> (<year>2005</year>) <article-title>Comprehensive Antibody Epitope Mapping of the Nucleocapsid Protein
of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Insight into
the Humoral Immunity of SARS</article-title>. <source>Clin. Chem.</source><volume>51</volume> (<issue>8</issue>), <fpage>1382</fpage>–<lpage>1396</lpage>. <pub-id pub-id-type="doi">10.1373/clinchem.2005.051045</pub-id>.<pub-id pub-id-type="pmid">15976093</pub-id></mixed-citation></ref><ref id="ref35"><mixed-citation publication-type="journal" id="cit35"><name><surname>Hu</surname><given-names>H.</given-names></name>; <name><surname>Li</surname><given-names>L.</given-names></name>; <name><surname>Kao</surname><given-names>R. Y.</given-names></name>; <name><surname>Kou</surname><given-names>B.</given-names></name>; <name><surname>Wang</surname><given-names>Z.</given-names></name>; et al. (<year>2005</year>) <article-title>Screening
and identification of linear B-cell epitopes and entry-blocking peptide
of severe acute respiratory syndrome (SARS)-associated coronavirus
using synthetic overlapping peptide library</article-title>. <source>J. Comb. Chem.</source><volume>7</volume>, <fpage>648</fpage>–<lpage>656</lpage>. <pub-id pub-id-type="doi">10.1021/cc0500607</pub-id>.<pub-id pub-id-type="pmid">16153058</pub-id></mixed-citation></ref><ref id="ref36"><mixed-citation publication-type="journal" id="cit36"><name><surname>Tam</surname><given-names>J. P.</given-names></name> (<year>1988</year>) <article-title>Synthetic
peptide vaccine design: synthesis and properties of a high-density
multiple antigenic peptide system</article-title>. <source>Proc. Natl.
Acad. Sci. U.S.A.</source><volume>85</volume>, <fpage>5409</fpage>–<lpage>5413</lpage>. <pub-id pub-id-type="doi">10.1073/pnas.85.15.5409</pub-id>.<pub-id pub-id-type="pmid">3399498</pub-id></mixed-citation></ref><ref id="ref37"><mixed-citation publication-type="journal" id="cit37"><name><surname>Qin</surname><given-names>C.</given-names></name>; <name><surname>Wang</surname><given-names>J.</given-names></name>; <name><surname>Wei</surname><given-names>Q.</given-names></name>; <name><surname>She</surname><given-names>M.</given-names></name>; <name><surname>Marasco</surname><given-names>W. A.</given-names></name>; et al. (<year>2005</year>) <article-title>An animal model of SARS produced by infection of Macaca mulatta with
SARS coronavirus</article-title>. <source>J. Pathol.</source><volume>206</volume>, <fpage>251</fpage>–<lpage>259</lpage>. <pub-id pub-id-type="doi">10.1002/path.1769</pub-id>.<pub-id pub-id-type="pmid">15892035</pub-id></mixed-citation></ref><ref id="ref38"><mixed-citation publication-type="journal" id="cit38"><name><surname>Wong</surname><given-names>S. K.</given-names></name>; <name><surname>Li</surname><given-names>W.</given-names></name>; <name><surname>Moore</surname><given-names>M. J.</given-names></name>; <name><surname>Choe</surname><given-names>H.</given-names></name>; <name><surname>Farzan</surname><given-names>M.</given-names></name> (<year>2003</year>) <article-title>A 193-amino acid fragment of the
SARS coronavirus S protein efficiently binds angiotensin-converting
enzyme 2</article-title>. <source>J. Biol. Chem.</source><volume>279</volume>, <fpage>3197</fpage>–<lpage>3201</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.C300520200</pub-id>.<pub-id pub-id-type="pmid">14670965</pub-id></mixed-citation></ref><ref id="ref39"><mixed-citation publication-type="journal" id="cit39"><name><surname>Li</surname><given-names>W.</given-names></name>; <name><surname>Zhang</surname><given-names>C.</given-names></name>; <name><surname>Sui</surname><given-names>J.</given-names></name>; <name><surname>Kuhn</surname><given-names>J. H.</given-names></name>; <name><surname>Moore</surname><given-names>M. J.</given-names></name>; et al. (<year>2005</year>) <article-title>Receptor and viral determinants
of SARS-coronavirus adaptation to human ACE2</article-title>. <source>EMBO J.</source><volume>24</volume>, <fpage>1634</fpage>–<lpage>1643</lpage>. <pub-id pub-id-type="doi">10.1038/sj.emboj.7600640</pub-id>.<pub-id pub-id-type="pmid">15791205</pub-id></mixed-citation></ref><ref id="ref40"><mixed-citation publication-type="journal" id="cit40"><name><surname>Xu</surname><given-names>Y.</given-names></name>; <name><surname>Zhu</surname><given-names>J.</given-names></name>; <name><surname>Liu</surname><given-names>Y.</given-names></name>; <name><surname>Lou</surname><given-names>Z.</given-names></name>; <name><surname>Yuan</surname><given-names>F.</given-names></name>; et al. (<year>2004</year>) <article-title>Characterization of
the heptad repeat regions, HR1 and HR2, and design of a fusion core
structure model of the spike protein from severe acute respiratory
syndrome (SARS) coronavirus</article-title>. <source>Biochemistry</source><volume>43</volume>, <fpage>14064</fpage>–<lpage>14071</lpage>. <pub-id pub-id-type="doi">10.1021/bi049101q</pub-id>.<pub-id pub-id-type="pmid">15518555</pub-id></mixed-citation></ref><ref id="ref41"><mixed-citation publication-type="journal" id="cit41"><name><surname>Xu</surname><given-names>Y.</given-names></name>; <name><surname>Lou</surname><given-names>Z.</given-names></name>; <name><surname>Liu</surname><given-names>Y.</given-names></name>; <name><surname>Pang</surname><given-names>H.</given-names></name>; <name><surname>Tien</surname><given-names>P.</given-names></name>; et al. (<year>2004</year>) <article-title>Crystal structure of
severe acute respiratory syndrome coronavirus spike protein fusion
core</article-title>. <source>J. Biol. Chem.</source><volume>279</volume>, <fpage>49414</fpage>–<lpage>49419</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.M408782200</pub-id>.<pub-id pub-id-type="pmid">15345712</pub-id></mixed-citation></ref><ref id="ref42"><mixed-citation publication-type="journal" id="cit42"><name><surname>Wang</surname><given-names>S. F.</given-names></name>; <name><surname>Tseng</surname><given-names>S. P.</given-names></name>; <name><surname>Yen</surname><given-names>C. H.</given-names></name>; <name><surname>Yang</surname><given-names>J. Y.</given-names></name>; <name><surname>Tsao</surname><given-names>C. H.</given-names></name>; <name><surname>Shen</surname><given-names>C. W.</given-names></name>; <name><surname>Chen</surname><given-names>K. H.</given-names></name>; <name><surname>Liu</surname><given-names>F. T.</given-names></name>; <name><surname>Liu</surname><given-names>W. T.</given-names></name>; <name><surname>Chen</surname><given-names>Y. M.</given-names></name>; <name><surname>Huang</surname><given-names>J. C.</given-names></name> (<year>2014</year>) <article-title>Antibody-dependent
SARS coronavirus infection is mediated by antibodies against spike
proteins</article-title>. <source>Biochem. Biophys. Res. Commun.</source><volume>451</volume> (<issue>2</issue>), <fpage>208</fpage>–<lpage>214</lpage>. <pub-id pub-id-type="doi">10.1016/j.bbrc.2014.07.090</pub-id>.<pub-id pub-id-type="pmid">25073113</pub-id></mixed-citation></ref><ref id="ref43"><mixed-citation publication-type="journal" id="cit43"><name><surname>Yip</surname><given-names>M. S.</given-names></name>; <name><surname>Leung</surname><given-names>N. H.</given-names></name>; <name><surname>Cheung</surname><given-names>C. Y.</given-names></name>; <name><surname>Li</surname><given-names>P. H.</given-names></name>; <name><surname>Lee</surname><given-names>H. H.</given-names></name>; <name><surname>Daëron</surname><given-names>M.</given-names></name>; <name><surname>Peiris</surname><given-names>J. S.</given-names></name>; <name><surname>Bruzzone</surname><given-names>R.</given-names></name>; <name><surname>Jaume</surname><given-names>M.</given-names></name> (<year>2014</year>) <article-title>Antibody-dependent
infection of human macrophages by severe acute respiratory syndrome
coronavirus</article-title>. <source>Virol. J.</source><volume>11</volume>, <fpage>82</fpage><pub-id pub-id-type="doi">10.1186/1743-422X-11-82</pub-id>.<pub-id pub-id-type="pmid">24885320</pub-id></mixed-citation></ref><ref id="ref44"><mixed-citation publication-type="journal" id="cit44"><name><surname>Jaume</surname><given-names>M.</given-names></name>; <name><surname>Yip</surname><given-names>M. S.</given-names></name>; <name><surname>Cheung</surname><given-names>C. Y.</given-names></name>; <name><surname>Leung</surname><given-names>H. L.</given-names></name>; <name><surname>Li</surname><given-names>P. H.</given-names></name>; <name><surname>Kien</surname><given-names>F.</given-names></name>; <name><surname>Dutry</surname><given-names>I.</given-names></name>; <name><surname>Callendret</surname><given-names>B.</given-names></name>; <name><surname>Escriou</surname><given-names>N.</given-names></name>; <name><surname>Altmeyer</surname><given-names>R.</given-names></name>; <name><surname>Nal</surname><given-names>B.</given-names></name>; <name><surname>Daëron</surname><given-names>M.</given-names></name>; <name><surname>Bruzzone</surname><given-names>R.</given-names></name>; <name><surname>Peiris</surname><given-names>J. S.</given-names></name> (<year>2011</year>) <article-title>Anti-Severe Acute Respiratory Syndrome
Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells
via a pH- and Cysteine Protease-Independent FcγR Pathwa</article-title>. <source>J. Virol.</source><volume>85</volume> (<issue>20</issue>), <fpage>10582</fpage>–<lpage>10597</lpage>. <pub-id pub-id-type="doi">10.1128/JVI.00671-11</pub-id>.<pub-id pub-id-type="pmid">21775467</pub-id></mixed-citation></ref><ref id="ref45"><mixed-citation publication-type="journal" id="cit45"><name><surname>Dawson</surname><given-names>P. E.</given-names></name>; <name><surname>Muir</surname><given-names>T. W.</given-names></name>; <name><surname>Clark-Lewis</surname><given-names>I.</given-names></name>; <name><surname>Kent</surname><given-names>S. B.</given-names></name> (<year>1994</year>) <article-title>Synthesis of proteins by native chemical ligation</article-title>. <source>Science</source><volume>266</volume>, <fpage>776</fpage>–<lpage>779</lpage>. <pub-id pub-id-type="doi">10.1126/science.7973629</pub-id>.<pub-id pub-id-type="pmid">7973629</pub-id></mixed-citation></ref><ref id="ref46"><mixed-citation publication-type="journal" id="cit46"><name><surname>Ruan</surname><given-names>Y. J.</given-names></name>; <name><surname>Wei</surname><given-names>C. L.</given-names></name>; <name><surname>Ee</surname><given-names>A. L.</given-names></name>; <name><surname>Vega</surname><given-names>V. B.</given-names></name>; <name><surname>Thoreau</surname><given-names>H.</given-names></name>; <name><surname>Su</surname><given-names>S. T.</given-names></name>; <name><surname>Chia</surname><given-names>J. M.</given-names></name>; <name><surname>Ng</surname><given-names>P.</given-names></name>; <name><surname>Chiu</surname><given-names>K. P.</given-names></name>; <name><surname>Lim</surname><given-names>L.</given-names></name>; <name><surname>Zhang</surname><given-names>T.</given-names></name>; <name><surname>Peng</surname><given-names>C. K.</given-names></name>; <name><surname>Lin</surname><given-names>E. O.</given-names></name>; <name><surname>Lee</surname><given-names>N. M.</given-names></name>; <name><surname>Yee</surname><given-names>S. L.</given-names></name>; <name><surname>Ng</surname><given-names>L. F.</given-names></name>; <name><surname>Chee</surname><given-names>R. E.</given-names></name>; <name><surname>Stanton</surname><given-names>L. W.</given-names></name>; <name><surname>Long</surname><given-names>P. M.</given-names></name>; <name><surname>Liu</surname><given-names>E. T.</given-names></name> (<year>2003</year>) <article-title>Comparative full-length genome sequence
analysis of 14 SARS coronavirus isolates and common mutations associated
with putative origins of infection</article-title>. <source>Lancet</source><volume>361</volume> (<issue>9371</issue>), <fpage>1779</fpage>–<lpage>1785</lpage>. <pub-id pub-id-type="doi">10.1016/S0140-6736(03)13414-9</pub-id>.<pub-id pub-id-type="pmid">12781537</pub-id></mixed-citation></ref><ref id="ref47"><mixed-citation publication-type="journal" id="cit47"><name><surname>Sakaguchi</surname><given-names>N.</given-names></name>; <name><surname>Kimura</surname><given-names>T.</given-names></name>; <name><surname>Matsushita</surname><given-names>S.</given-names></name>; <name><surname>Fujimura</surname><given-names>S.</given-names></name>; <name><surname>Shibata</surname><given-names>J.</given-names></name>; et al. (<year>2005</year>) <article-title>Generation of High-Affinity Antibody against T Cell-Dependent Antigen
in the Ganp Gene-Transgenic Mouse</article-title>. <source>J. Immunol.</source><volume>174</volume>, <fpage>4485</fpage>–<lpage>4494</lpage>. <pub-id pub-id-type="doi">10.4049/jimmunol.174.8.4485</pub-id>.<pub-id pub-id-type="pmid">15814669</pub-id></mixed-citation></ref><ref id="ref48"><mixed-citation publication-type="journal" id="cit48"><name><surname>Kuwahara</surname><given-names>K.</given-names></name>; <name><surname>Yoshida</surname><given-names>M.</given-names></name>; <name><surname>Kondo</surname><given-names>E.</given-names></name>; <name><surname>Sakata</surname><given-names>A.</given-names></name>; <name><surname>Watanabe</surname><given-names>Y.</given-names></name>; et al. (<year>2000</year>) <article-title>A novel nuclear phosphoprotein,
GANP, is up-regulated in centrocytes of the germinal center and associated
with MCM3, a protein essential for DNA replication</article-title>. <source>Blood</source><volume>95</volume>, <fpage>2321</fpage>–<lpage>2328</lpage>.<pub-id pub-id-type="pmid">10733502</pub-id></mixed-citation></ref><ref id="ref49"><mixed-citation publication-type="journal" id="cit49"><name><surname>Jönsson</surname><given-names>U.</given-names></name> (<year>1991</year>) <article-title>Real-time biospecific
interaction analysis using surface plasmon resonance and sensor chip
technology</article-title>. <source>BioTechniques</source><volume>11</volume>, <fpage>620</fpage>–<lpage>627</lpage>.<pub-id pub-id-type="pmid">1804254</pub-id></mixed-citation></ref><ref id="ref50"><mixed-citation publication-type="journal" id="cit50"><name><surname>Johnsson</surname><given-names>B.</given-names></name>; <name><surname>Löfas</surname><given-names>S.</given-names></name>; <name><surname>Lindquist</surname><given-names>G.</given-names></name> (<year>1991</year>) <article-title>Immobilization
of proteins to a carboxymethyldextran-modified gold surface for biospecific
interaction analysis in surface plasmon resonance sensors</article-title>. <source>Anal. Biochem.</source><volume>198</volume>, <fpage>268</fpage>–<lpage>277</lpage>. <pub-id pub-id-type="doi">10.1016/0003-2697(91)90424-R</pub-id>.<pub-id pub-id-type="pmid">1724720</pub-id></mixed-citation></ref><ref id="ref51"><mixed-citation publication-type="journal" id="cit51"><name><surname>Karlsson</surname><given-names>R.</given-names></name>; <name><surname>Michaelsson</surname><given-names>A.</given-names></name>; <name><surname>Mattsson</surname><given-names>L.</given-names></name> (<year>1991</year>) <article-title>Kinetic analysis of
monoclonal antibody-antigen interactions with a new biosensor based
analytical system</article-title>. <source>J. Immunol. Methods</source><volume>145</volume>, <fpage>229</fpage>–<lpage>240</lpage>. <pub-id pub-id-type="doi">10.1016/0022-1759(91)90331-9</pub-id>.<pub-id pub-id-type="pmid">1765656</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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