Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">HLA-A<sup>∗</sup>0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins</title><author><name sortKey="Tsao, Yeou Ping" sort="Tsao, Yeou Ping" uniqKey="Tsao Y" first="Yeou-Ping" last="Tsao">Yeou-Ping Tsao</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Lin, Jian Yu" sort="Lin, Jian Yu" uniqKey="Lin J" first="Jian-Yu" last="Lin">Jian-Yu Lin</name><affiliation><nlm:aff id="aff1">Department of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Jan, Jia Tsrong" sort="Jan, Jia Tsrong" uniqKey="Jan J" first="Jia-Tsrong" last="Jan">Jia-Tsrong Jan</name><affiliation><nlm:aff id="aff2">Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Leng, Chih Hsiang" sort="Leng, Chih Hsiang" uniqKey="Leng C" first="Chih-Hsiang" last="Leng">Chih-Hsiang Leng</name><affiliation><nlm:aff id="aff3">Vaccine Research and Development Center, National Health Research Institutes, Taipei 115, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chu, Chen Chung" sort="Chu, Chen Chung" uniqKey="Chu C" first="Chen-Chung" last="Chu">Chen-Chung Chu</name><affiliation><nlm:aff id="aff5">Department of Medical Research, Mackay Memorial Hospital, Taipei 104, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Yang, Yuh Cheng" sort="Yang, Yuh Cheng" uniqKey="Yang Y" first="Yuh-Cheng" last="Yang">Yuh-Cheng Yang</name><affiliation><nlm:aff id="aff5">Department of Medical Research, Mackay Memorial Hospital, Taipei 104, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chen, Show Li" sort="Chen, Show Li" uniqKey="Chen S" first="Show-Li" last="Chen">Show-Li Chen</name><affiliation><nlm:aff id="aff1">Department of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan</nlm:aff></affiliation><affiliation><nlm:aff id="aff6">Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">16630549</idno><idno type="pmc">7092919</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092919</idno><idno type="RBID">PMC:7092919</idno><idno type="doi">10.1016/j.bbrc.2006.03.152</idno><date when="2006">2006</date><idno type="wicri:Area/Pmc/Corpus">000E28</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000E28</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">HLA-A<sup>∗</sup>0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins</title><author><name sortKey="Tsao, Yeou Ping" sort="Tsao, Yeou Ping" uniqKey="Tsao Y" first="Yeou-Ping" last="Tsao">Yeou-Ping Tsao</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Lin, Jian Yu" sort="Lin, Jian Yu" uniqKey="Lin J" first="Jian-Yu" last="Lin">Jian-Yu Lin</name><affiliation><nlm:aff id="aff1">Department of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Jan, Jia Tsrong" sort="Jan, Jia Tsrong" uniqKey="Jan J" first="Jia-Tsrong" last="Jan">Jia-Tsrong Jan</name><affiliation><nlm:aff id="aff2">Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Leng, Chih Hsiang" sort="Leng, Chih Hsiang" uniqKey="Leng C" first="Chih-Hsiang" last="Leng">Chih-Hsiang Leng</name><affiliation><nlm:aff id="aff3">Vaccine Research and Development Center, National Health Research Institutes, Taipei 115, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chu, Chen Chung" sort="Chu, Chen Chung" uniqKey="Chu C" first="Chen-Chung" last="Chu">Chen-Chung Chu</name><affiliation><nlm:aff id="aff5">Department of Medical Research, Mackay Memorial Hospital, Taipei 104, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Yang, Yuh Cheng" sort="Yang, Yuh Cheng" uniqKey="Yang Y" first="Yuh-Cheng" last="Yang">Yuh-Cheng Yang</name><affiliation><nlm:aff id="aff5">Department of Medical Research, Mackay Memorial Hospital, Taipei 104, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Chen, Show Li" sort="Chen, Show Li" uniqKey="Chen S" first="Show-Li" last="Chen">Show-Li Chen</name><affiliation><nlm:aff id="aff1">Department of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan</nlm:aff></affiliation><affiliation><nlm:aff id="aff6">Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan</nlm:aff></affiliation></author></analytic><series><title level="j">Biochemical and Biophysical Research Communications</title><idno type="ISSN">0006-291X</idno><idno type="eISSN">1090-2104</idno><imprint><date when="2006">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The immunogenicity of HLA-A<sup>∗</sup>0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins’ ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (<ext-link ext-link-type="uri" xlink:href="http://thr.cit.nih.gov/molbio/hla_bind/">http://thr.cit.nih.gov/molbio/hla_bind/</ext-link>), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223–231, N227–235, and N317–325 to be the first identified HLA-A<sup>∗</sup>0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A<sup>∗</sup>0201-restricted CTL epitopes of S protein (S978–986, S1203–1211, and S1167–1175), here we found two novel peptides S787–795 and S1042–1050 as S-specific CTL epitopes. Moreover, our identified N317–325 and S1042–1050 CTL epitopes could induce recall responses when IFN-γ stimulation of blood CD8<sup>+</sup> T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Ksiazek, G" uniqKey="Ksiazek G">G. Ksiazek</name></author><author><name sortKey="Endman, D" uniqKey="Endman D">D. Endman</name></author><author><name sortKey="Goldsmith, C S" uniqKey="Goldsmith C">C.S. Goldsmith</name></author><author><name sortKey="Zaki, S R" uniqKey="Zaki S">S.R. Zaki</name></author><author><name sortKey="Peret, T" uniqKey="Peret T">T. Peret</name></author><author><name sortKey="Emery, S" uniqKey="Emery S">S. Emery</name></author><author><name sortKey="Tong, S" uniqKey="Tong S">S. Tong</name></author><author><name sortKey="Urbani, C" uniqKey="Urbani C">C. Urbani</name></author><author><name sortKey="Comer, J A" uniqKey="Comer J">J.A. Comer</name></author><author><name sortKey="Lim, W" uniqKey="Lim W">W. Lim</name></author><author><name sortKey="Rollin, P E" uniqKey="Rollin P">P.E. Rollin</name></author><author><name sortKey="Dowell, S F" uniqKey="Dowell S">S.F. Dowell</name></author><author><name sortKey="Ling, A E" uniqKey="Ling A">A.E. Ling</name></author><author><name sortKey="Humphrey, C D" uniqKey="Humphrey C">C.D. Humphrey</name></author><author><name sortKey="Shieh, W J" uniqKey="Shieh W">W.J. Shieh</name></author><author><name sortKey="Guarner, J" uniqKey="Guarner J">J. Guarner</name></author><author><name sortKey="Paddock, C D" uniqKey="Paddock C">C.D. Paddock</name></author><author><name sortKey="Rota, P" uniqKey="Rota P">P. Rota</name></author><author><name sortKey="Fields, B" uniqKey="Fields B">B. Fields</name></author><author><name sortKey="Derisi, J" uniqKey="Derisi J">J. DeRisi</name></author><author><name sortKey="Yang, J Y" uniqKey="Yang J">J.Y. Yang</name></author><author><name sortKey="Cox, N" uniqKey="Cox N">N. Cox</name></author><author><name sortKey="Hughes, J M" uniqKey="Hughes J">J.M. Hughes</name></author><author><name sortKey="Leduc, J W" uniqKey="Leduc J">J.W. LeDuc</name></author><author><name sortKey="Bellini, W J" uniqKey="Bellini W">W.J. Bellini</name></author><author><name sortKey="Anderson, L J" uniqKey="Anderson L">L.J. Anderson</name></author><author><name sortKey="Group, S W" uniqKey="Group S">S.W. Group</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Poutanen, M" uniqKey="Poutanen M">M. Poutanen</name></author><author><name sortKey="Low, E" uniqKey="Low E">E. Low</name></author><author><name sortKey="Henry, B" uniqKey="Henry B">B. Henry</name></author><author><name sortKey="Finkelstein, S" uniqKey="Finkelstein S">S. Finkelstein</name></author><author><name sortKey="Rose, D" uniqKey="Rose D">D. Rose</name></author><author><name sortKey="Green, K" uniqKey="Green K">K. Green</name></author><author><name sortKey="Tellier, R" uniqKey="Tellier R">R. Tellier</name></author><author><name sortKey="Draker, R" uniqKey="Draker R">R. Draker</name></author><author><name sortKey="Adachi, D" uniqKey="Adachi D">D. Adachi</name></author><author><name sortKey="Ayers, M" uniqKey="Ayers M">M. Ayers</name></author><author><name sortKey="Chan, A K" uniqKey="Chan A">A.K. Chan</name></author><author><name sortKey="Skowronski, D M" uniqKey="Skowronski D">D.M. Skowronski</name></author><author><name sortKey="Salit, I" uniqKey="Salit I">I. Salit</name></author><author><name sortKey="Simor, A E" uniqKey="Simor A">A.E. Simor</name></author><author><name sortKey="Slutsky, A S" uniqKey="Slutsky A">A.S. Slutsky</name></author><author><name sortKey="Doyle, P W" uniqKey="Doyle P">P.W. Doyle</name></author><author><name sortKey="Krajden, M" uniqKey="Krajden M">M. Krajden</name></author><author><name sortKey="Petric, M" uniqKey="Petric M">M. Petric</name></author><author><name sortKey="Brunham, R C" uniqKey="Brunham R">R.C. Brunham</name></author><author><name sortKey="Mcgeer, A J" uniqKey="Mcgeer A">A.J. McGeer</name></author><author><name sortKey="C National Microbiology Laboratory" uniqKey="C National Microbiology Laboratory">C. National Microbiology Laboratory</name></author><author><name sortKey="C S A R S S Team" uniqKey="C S A R S S Team">C. S. A. R. S. S. Team</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, L A" uniqKey="Ee L">L.A. 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="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><author><name sortKey="Berne, M A" uniqKey="Berne M">M.A. Berne</name></author><author><name sortKey="Somasundaran, M" uniqKey="Somasundaran M">M. Somasundaran</name></author><author><name sortKey="Sullivan, J L" uniqKey="Sullivan J">J.L. Sullivan</name></author><author><name sortKey="Luzuriaga, K" uniqKey="Luzuriaga K">K. Luzuriaga</name></author><author><name sortKey="Greenough, T C" uniqKey="Greenough T">T.C. Greenough</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="Pei, J" uniqKey="Pei J">J. Pei</name></author><author><name sortKey="Briles, W E" uniqKey="Briles W">W.E. Briles</name></author><author><name sortKey="Collisson, E W" uniqKey="Collisson E">E.W. Collisson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Seo, S H" uniqKey="Seo S">S.H. Seo</name></author><author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name></author><author><name sortKey="Smith, R" uniqKey="Smith R">R. Smith</name></author><author><name sortKey="Collisson, E W" uniqKey="Collisson E">E.W. Collisson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ressing, M E" uniqKey="Ressing M">M.E. Ressing</name></author><author><name sortKey="Van Driel, W J" uniqKey="Van Driel W">W.J. van Driel</name></author><author><name sortKey="Celis, E" uniqKey="Celis E">E. Celis</name></author><author><name sortKey="Sette, A" uniqKey="Sette A">A. Sette</name></author><author><name sortKey="Brandt, M P" uniqKey="Brandt M">M.P. Brandt</name></author><author><name sortKey="Hartman, M" uniqKey="Hartman M">M. Hartman</name></author><author><name sortKey="Anholts, J D" uniqKey="Anholts J">J.D. Anholts</name></author><author><name sortKey="Schreuder, G M" uniqKey="Schreuder G">G.M. Schreuder</name></author><author><name sortKey="Ter Harmsel, W B" uniqKey="Ter Harmsel W">W.B. ter Harmsel</name></author><author><name sortKey="Fleuren, G J" uniqKey="Fleuren G">G.J. Fleuren</name></author><author><name sortKey="Trimbos, B J" uniqKey="Trimbos B">B.J. Trimbos</name></author><author><name sortKey="Kast, W M" uniqKey="Kast W">W.M. Kast</name></author><author><name sortKey="Melief, C J" uniqKey="Melief C">C.J. Melief</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Passoni, L" uniqKey="Passoni L">L. Passoni</name></author><author><name sortKey="Scardino, A" uniqKey="Scardino A">A. Scardino</name></author><author><name sortKey="Bertazzoli, C" uniqKey="Bertazzoli C">C. Bertazzoli</name></author><author><name sortKey="Gallo, B" uniqKey="Gallo B">B. Gallo</name></author><author><name sortKey="Coluccia, A M" uniqKey="Coluccia A">A.M. Coluccia</name></author><author><name sortKey="Lemonnier, F A" uniqKey="Lemonnier F">F.A. Lemonnier</name></author><author><name sortKey="Kosmatopoulos, K" uniqKey="Kosmatopoulos K">K. Kosmatopoulos</name></author><author><name sortKey="Gambacorti Passerini, C" uniqKey="Gambacorti Passerini C">C. Gambacorti-Passerini</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wolfel, T" uniqKey="Wolfel T">T. Wolfel</name></author><author><name sortKey="Van Pel, A" uniqKey="Van Pel A">A. Van Pel</name></author><author><name sortKey="Brichard, V" uniqKey="Brichard V">V. Brichard</name></author><author><name sortKey="Schneider, J" uniqKey="Schneider J">J. Schneider</name></author><author><name sortKey="Seliger, B" uniqKey="Seliger B">B. Seliger</name></author><author><name sortKey="Meyer Zum Buschenfelde, K H" uniqKey="Meyer Zum Buschenfelde K">K.H. Meyer zum Buschenfelde</name></author><author><name sortKey="Boon, T" uniqKey="Boon T">T. Boon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, S J" uniqKey="Liu S">S.J. Liu</name></author><author><name sortKey="Leng, C H" uniqKey="Leng C">C.H. Leng</name></author><author><name sortKey="Lien, S P" uniqKey="Lien S">S.P. Lien</name></author><author><name sortKey="Chi, H Y" uniqKey="Chi H">H.Y. Chi</name></author><author><name sortKey="Huang, C Y" uniqKey="Huang C">C.Y. Huang</name></author><author><name sortKey="Lin, C L" uniqKey="Lin C">C.L. Lin.</name></author><author><name sortKey="Lian, W C" uniqKey="Lian W">W.C. Lian</name></author><author><name sortKey="Chen, C J" uniqKey="Chen C">C.J. Chen</name></author><author><name sortKey="Hsieh, S L" uniqKey="Hsieh S">S.L. Hsieh</name></author><author><name sortKey="Chong, P" uniqKey="Chong P">P. Chong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bullock, T N" uniqKey="Bullock T">T.N. Bullock</name></author><author><name sortKey="Colella, T A" uniqKey="Colella T">T.A. Colella</name></author><author><name sortKey="Engelhard, V H" uniqKey="Engelhard V">V.H. Engelhard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lin, C W" uniqKey="Lin C">C.W. Lin</name></author><author><name sortKey="Lee, J Y" uniqKey="Lee J">J.Y. Lee</name></author><author><name sortKey="Tsao, Y P" uniqKey="Tsao Y">Y.P. Tsao</name></author><author><name sortKey="Shen, C P" uniqKey="Shen C">C.P. Shen</name></author><author><name sortKey="Lai, H C" uniqKey="Lai H">H.C. Lai</name></author><author><name sortKey="Chen, S L" uniqKey="Chen S">S.L. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Hou, J" uniqKey="Hou J">J. Hou</name></author><author><name sortKey="Jiang, X" uniqKey="Jiang X">X. Jiang</name></author><author><name sortKey="Ma, S" uniqKey="Ma S">S. Ma</name></author><author><name sortKey="Meng, M" uniqKey="Meng M">M. Meng</name></author><author><name sortKey="Wang, B" uniqKey="Wang B">B. Wang</name></author><author><name sortKey="Zhang, M" uniqKey="Zhang M">M. Zhang</name></author><author><name sortKey="Zhang, M" uniqKey="Zhang M">M. Zhang</name></author><author><name sortKey="Tang, X" uniqKey="Tang X">X. Tang</name></author><author><name sortKey="Zhang, F" uniqKey="Zhang F">F. Zhang</name></author><author><name sortKey="Wan, T" uniqKey="Wan T">T. Wan</name></author><author><name sortKey="Li, N" uniqKey="Li N">N. Li</name></author><author><name sortKey="Yu, Y" uniqKey="Yu Y">Y. Yu</name></author><author><name sortKey="Hu, H" uniqKey="Hu H">H. Hu</name></author><author><name sortKey="Yang, R" uniqKey="Yang R">R. Yang</name></author><author><name sortKey="He, W" uniqKey="He W">W. He</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X. Wang</name></author><author><name sortKey="Cao, X" uniqKey="Cao X">X. Cao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lin, M" uniqKey="Lin M">M. Lin</name></author><author><name sortKey="Chu, C C" uniqKey="Chu C">C.C. Chu</name></author><author><name sortKey="Lee, H L" uniqKey="Lee H">H.L. Lee</name></author><author><name sortKey="Chang, S L" uniqKey="Chang S">S.L. Chang</name></author><author><name sortKey="Ohashi, J" uniqKey="Ohashi J">J. Ohashi</name></author><author><name sortKey="Tokunaga, K" uniqKey="Tokunaga K">K. Tokunaga</name></author><author><name sortKey="Akaza, T" uniqKey="Akaza T">T. Akaza</name></author><author><name sortKey="Juji, T" uniqKey="Juji T">T. Juji</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kaufmann, A M" uniqKey="Kaufmann A">A.M. Kaufmann</name></author><author><name sortKey="Nieland, J" uniqKey="Nieland J">J. Nieland</name></author><author><name sortKey="Schinz, M" uniqKey="Schinz M">M. Schinz</name></author><author><name sortKey="Nonn, M" uniqKey="Nonn M">M. Nonn</name></author><author><name sortKey="Gabelsberger, J" uniqKey="Gabelsberger J">J. Gabelsberger</name></author><author><name sortKey="Meissner, H" uniqKey="Meissner H">H. Meissner</name></author><author><name sortKey="Muller, R T" uniqKey="Muller R">R.T. Muller</name></author><author><name sortKey="Jochmus, I" uniqKey="Jochmus I">I. Jochmus</name></author><author><name sortKey="Gissmann, L" uniqKey="Gissmann L">L. Gissmann</name></author><author><name sortKey="Schneider, A" uniqKey="Schneider A">A. Schneider</name></author><author><name sortKey="Durst, M" uniqKey="Durst M">M. Durst</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hsueh, P R" uniqKey="Hsueh P">P.R. Hsueh</name></author><author><name sortKey="Yang, P C" uniqKey="Yang P">P.C. Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vigneau, S" uniqKey="Vigneau S">S. Vigneau</name></author><author><name sortKey="Rohrlich, P S" uniqKey="Rohrlich P">P.S. Rohrlich</name></author><author><name sortKey="Brahic, M" uniqKey="Brahic M">M. Brahic</name></author><author><name sortKey="Bureau, J F" uniqKey="Bureau J">J.F. Bureau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, Y F" uniqKey="Chen Y">Y.F. Chen</name></author><author><name sortKey="Lin, C W" uniqKey="Lin C">C.W. Lin</name></author><author><name sortKey="Tsao, Y P" uniqKey="Tsao Y">Y.P. Tsao</name></author><author><name sortKey="Chen, S L" uniqKey="Chen S">S.L. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Evans, C" uniqKey="Evans C">C. Evans</name></author><author><name sortKey="Bauer, S" uniqKey="Bauer S">S. Bauer</name></author><author><name sortKey="Grubert, T" uniqKey="Grubert T">T. Grubert</name></author><author><name sortKey="Brucker, C" uniqKey="Brucker C">C. Brucker</name></author><author><name sortKey="Baur, S" uniqKey="Baur S">S. Baur</name></author><author><name sortKey="Heeg, K" uniqKey="Heeg K">K. Heeg</name></author><author><name sortKey="Wagner, H" uniqKey="Wagner H">H. Wagner</name></author><author><name sortKey="Lipford, G B" uniqKey="Lipford G">G.B. Lipford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nakagawa, M" uniqKey="Nakagawa M">M. Nakagawa</name></author><author><name sortKey="Stites, D P" uniqKey="Stites D">D.P. Stites</name></author><author><name sortKey="Farhat, S" uniqKey="Farhat S">S. Farhat</name></author><author><name sortKey="Sisler, J R" uniqKey="Sisler J">J.R. Sisler</name></author><author><name sortKey="Moss, B" uniqKey="Moss B">B. Moss</name></author><author><name sortKey="Kong, F" uniqKey="Kong F">F. Kong</name></author><author><name sortKey="Moscicki, A B" uniqKey="Moscicki A">A.B. Moscicki</name></author><author><name sortKey="Palefsky, J M" uniqKey="Palefsky J">J.M. Palefsky</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Borysiewicz, L K" uniqKey="Borysiewicz L">L.K. Borysiewicz</name></author><author><name sortKey="Fiander, A" uniqKey="Fiander A">A. Fiander</name></author><author><name sortKey="Nimako, M" uniqKey="Nimako M">M. Nimako</name></author><author><name sortKey="Man, S" uniqKey="Man S">S. Man</name></author><author><name sortKey="Wilkinson, G W" uniqKey="Wilkinson G">G.W. Wilkinson</name></author><author><name sortKey="Westmoreland, D" uniqKey="Westmoreland D">D. Westmoreland</name></author><author><name sortKey="Evans, A S" uniqKey="Evans A">A.S. Evans</name></author><author><name sortKey="Adams, M" uniqKey="Adams M">M. Adams</name></author><author><name sortKey="Stacey, S N" uniqKey="Stacey S">S.N. Stacey</name></author><author><name sortKey="Boursnell, M E" uniqKey="Boursnell M">M.E. Boursnell</name></author><author><name sortKey="Rutherford, E" uniqKey="Rutherford E">E. Rutherford</name></author><author><name sortKey="Hickling, J K" uniqKey="Hickling J">J.K. Hickling</name></author><author><name sortKey="Inglis, S C" uniqKey="Inglis S">S.C. Inglis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schreurs, M W" uniqKey="Schreurs M">M.W. Schreurs</name></author><author><name sortKey="Scholten, K B" uniqKey="Scholten K">K.B. Scholten</name></author><author><name sortKey="Kueter, E W" uniqKey="Kueter E">E.W. Kueter</name></author><author><name sortKey="Ruizendaal, J J" uniqKey="Ruizendaal J">J.J. Ruizendaal</name></author><author><name sortKey="Meijer, C J" uniqKey="Meijer C">C.J. Meijer</name></author><author><name sortKey="Hooijberg, E" uniqKey="Hooijberg E">E. Hooijberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rivoltini, L" uniqKey="Rivoltini L">L. Rivoltini</name></author><author><name sortKey="Loftus, D J" uniqKey="Loftus D">D.J. Loftus</name></author><author><name sortKey="Barracchini, K" uniqKey="Barracchini K">K. Barracchini</name></author><author><name sortKey="Arienti, F" uniqKey="Arienti F">F. Arienti</name></author><author><name sortKey="Mazzocchi, A" uniqKey="Mazzocchi A">A. Mazzocchi</name></author><author><name sortKey="Biddison, W E" uniqKey="Biddison W">W.E. Biddison</name></author><author><name sortKey="Salgaller, M L" uniqKey="Salgaller M">M.L. Salgaller</name></author><author><name sortKey="Appella, E" uniqKey="Appella E">E. Appella</name></author><author><name sortKey="Parmiani, G" uniqKey="Parmiani G">G. Parmiani</name></author><author><name sortKey="Marincola, F M" uniqKey="Marincola F">F.M. Marincola</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><author><name sortKey="Li, N" uniqKey="Li N">N. Li</name></author><author><name sortKey="Zhou, X" uniqKey="Zhou X">X. Zhou</name></author><author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name></author><author><name sortKey="Yang, F" uniqKey="Yang F">F. Yang</name></author><author><name sortKey="Yu, Y" uniqKey="Yu Y">Y. Yu</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X. Wang</name></author><author><name sortKey="Yang, R" uniqKey="Yang R">R. Yang</name></author><author><name sortKey="Cao, X" uniqKey="Cao X">X. Cao</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><author><name sortKey="Pang, X W" uniqKey="Pang X">X.W. Pang</name></author><author><name sortKey="He, X Y" uniqKey="He X">X.Y. He</name></author><author><name sortKey="Zhang, H G" uniqKey="Zhang H">H.G. Zhang</name></author><author><name sortKey="Ng, J N" uniqKey="Ng J">J.N. Ng</name></author><author><name sortKey="Cheng, C S" uniqKey="Cheng C">C.S. Cheng</name></author><author><name sortKey="Yu, J" uniqKey="Yu J">J. Yu</name></author><author><name sortKey="Meng, L" uniqKey="Meng L">L. Meng</name></author><author><name sortKey="Yang, R F" uniqKey="Yang R">R.F. Yang</name></author><author><name sortKey="Lai, S T" uniqKey="Lai S">S.T. Lai</name></author><author><name sortKey="Guo, Z H" uniqKey="Guo Z">Z.H. Guo</name></author><author><name sortKey="Xie, Y" uniqKey="Xie Y">Y. Xie</name></author><author><name sortKey="Chen, W F" uniqKey="Chen W">W.F. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huang, L R" uniqKey="Huang L">L.R. Huang</name></author><author><name sortKey="Chiu, C M" uniqKey="Chiu C">C.M. Chiu</name></author><author><name sortKey="Yeh, S H" uniqKey="Yeh S">S.H. Yeh</name></author><author><name sortKey="Huang, W H" uniqKey="Huang W">W.H. Huang</name></author><author><name sortKey="Hsue, P R" uniqKey="Hsue P">P.R. Hsue</name></author><author><name sortKey="Yang, W Z" uniqKey="Yang W">W.Z. Yang</name></author><author><name sortKey="Yang, J Y" uniqKey="Yang J">J.Y. Yang</name></author><author><name sortKey="Su, I J" uniqKey="Su I">I.J. Su</name></author><author><name sortKey="Chang, S C" uniqKey="Chang S">S.C. Chang</name></author><author><name sortKey="Chen, P J" uniqKey="Chen P">P.J. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Leung, D T" uniqKey="Leung D">D.T. Leung</name></author><author><name sortKey="Tam, F C" uniqKey="Tam F">F.C. Tam</name></author><author><name sortKey="Ma, C H" uniqKey="Ma C">C.H. Ma</name></author><author><name sortKey="Chan, P K" uniqKey="Chan P">P.K. Chan</name></author><author><name sortKey="Cheung, J L" uniqKey="Cheung J">J.L. Cheung</name></author><author><name sortKey="Niu, H" uniqKey="Niu H">H. Niu</name></author><author><name sortKey="Tam, J S" uniqKey="Tam J">J.S. Tam</name></author><author><name sortKey="Lim, P L" uniqKey="Lim P">P.L. Lim</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Daginakatte, G C" uniqKey="Daginakatte G">G.C. Daginakatte</name></author><author><name sortKey="Chard Bergstrom, C" uniqKey="Chard Bergstrom C">C. Chard-Bergstrom</name></author><author><name sortKey="Andrews, G A" uniqKey="Andrews G">G.A. Andrews</name></author><author><name sortKey="Kapil, S" uniqKey="Kapil S">S. Kapil</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ndifuna, A" uniqKey="Ndifuna A">A. Ndifuna</name></author><author><name sortKey="Waters, A K" uniqKey="Waters A">A.K. Waters</name></author><author><name sortKey="Zhou, M" uniqKey="Zhou M">M. Zhou</name></author><author><name sortKey="Collisson, E W" uniqKey="Collisson E">E.W. Collisson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gao, W" uniqKey="Gao W">W. Gao</name></author><author><name sortKey="Tamin, A" uniqKey="Tamin A">A. Tamin</name></author><author><name sortKey="Soloff, A" uniqKey="Soloff A">A. Soloff</name></author><author><name sortKey="Aiuto, L D" uniqKey="Aiuto L">L.D. Aiuto</name></author><author><name sortKey="Nwanegbo, E" uniqKey="Nwanegbo E">E. Nwanegbo</name></author><author><name sortKey="Robbins, P D" uniqKey="Robbins P">P.D. Robbins</name></author><author><name sortKey="Bellini, W J" uniqKey="Bellini W">W.J. Bellini</name></author><author><name sortKey="Barratt Boyes, S" uniqKey="Barratt Boyes S">S. Barratt-Boyes</name></author><author><name sortKey="Gambotto, A" uniqKey="Gambotto A">A. Gambotto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jin, H" uniqKey="Jin H">H. Jin</name></author><author><name sortKey="Xiao, C" uniqKey="Xiao C">C. Xiao</name></author><author><name sortKey="Chen, Z" uniqKey="Chen Z">Z. Chen</name></author><author><name sortKey="Kang, Y" uniqKey="Kang Y">Y. Kang</name></author><author><name sortKey="Ma, Y" uniqKey="Ma Y">Y. Ma</name></author><author><name sortKey="Zhu, K" uniqKey="Zhu K">K. Zhu</name></author><author><name sortKey="Xie, Q" uniqKey="Xie Q">Q. Xie</name></author><author><name sortKey="Tu, Y" uniqKey="Tu Y">Y. Tu</name></author><author><name sortKey="Yu, Y" uniqKey="Yu Y">Y. Yu</name></author><author><name sortKey="Wang, B" uniqKey="Wang B">B. Wang</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><author><name sortKey="Icenogle, J P" uniqKey="Icenogle J">J.P. Icenogle</name></author><author><name sortKey="Penaranda, S" uniqKey="Penaranda S">S. Penaranda</name></author><author><name sortKey="Bankamp, B" uniqKey="Bankamp B">B. Bankamp</name></author><author><name sortKey="Maher, K" uniqKey="Maher K">K. Maher</name></author><author><name sortKey="Chen, M H" uniqKey="Chen M">M.H. Chen</name></author><author><name sortKey="Tong, S" uniqKey="Tong S">S. Tong</name></author><author><name sortKey="Tamin, A" uniqKey="Tamin A">A. Tamin</name></author><author><name sortKey="Lowe, L" uniqKey="Lowe L">L. Lowe</name></author><author><name sortKey="Frace, M" uniqKey="Frace M">M. Frace</name></author><author><name sortKey="Derisi, J L" uniqKey="Derisi J">J.L. DeRisi</name></author><author><name sortKey="Chen, Q" uniqKey="Chen Q">Q. Chen</name></author><author><name sortKey="Wang, D" uniqKey="Wang D">D. Wang</name></author><author><name sortKey="Erdman, D D" uniqKey="Erdman D">D.D. Erdman</name></author><author><name sortKey="Peret, T C" uniqKey="Peret T">T.C. Peret</name></author><author><name sortKey="Burns, C" uniqKey="Burns C">C. Burns</name></author><author><name sortKey="Ksiazek, T G" uniqKey="Ksiazek T">T.G. Ksiazek</name></author><author><name sortKey="Rollin, P E" uniqKey="Rollin P">P.E. Rollin</name></author><author><name sortKey="Sanchez, A" uniqKey="Sanchez A">A. Sanchez</name></author><author><name sortKey="Liffick, S" uniqKey="Liffick S">S. Liffick</name></author><author><name sortKey="Holloway, B" uniqKey="Holloway B">B. Holloway</name></author><author><name sortKey="Limor, J" uniqKey="Limor J">J. Limor</name></author><author><name sortKey="Mccaustland, K" uniqKey="Mccaustland K">K. McCaustland</name></author><author><name sortKey="Olsen Rasmussen, M" uniqKey="Olsen Rasmussen M">M. Olsen-Rasmussen</name></author><author><name sortKey="Fouchier, R" uniqKey="Fouchier R">R. Fouchier</name></author><author><name sortKey="Gunther, S" uniqKey="Gunther S">S. Gunther</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A.D. Osterhaus</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author><author><name sortKey="Pallansch, M A" uniqKey="Pallansch M">M.A. Pallansch</name></author><author><name sortKey="Anderson, L J" uniqKey="Anderson L">L.J. Anderson</name></author><author><name sortKey="Bellini, W J" uniqKey="Bellini W">W.J. Bellini</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Buchholz, U J" uniqKey="Buchholz U">U.J. Buchholz</name></author><author><name sortKey="Bukreyev, A" uniqKey="Bukreyev A">A. Bukreyev</name></author><author><name sortKey="Yang, L" uniqKey="Yang L">L. Yang</name></author><author><name sortKey="Lamirande, E W" uniqKey="Lamirande E">E.W. Lamirande</name></author><author><name sortKey="Murphy, B R" uniqKey="Murphy B">B.R. Murphy</name></author><author><name sortKey="Subbarao, K" uniqKey="Subbarao K">K. Subbarao</name></author><author><name sortKey="Collins, P L" uniqKey="Collins P">P.L. Collins</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="He, Y" uniqKey="He Y">Y. He</name></author><author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name></author><author><name sortKey="Liu, S" uniqKey="Liu S">S. Liu</name></author><author><name sortKey="Kou, Z" uniqKey="Kou Z">Z. Kou</name></author><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M. Farzan</name></author><author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sui, J" uniqKey="Sui J">J. Sui</name></author><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Murakami, A" uniqKey="Murakami A">A. Murakami</name></author><author><name sortKey="Tamin, A" uniqKey="Tamin A">A. Tamin</name></author><author><name sortKey="Matthews, L J" uniqKey="Matthews L">L.J. Matthews</name></author><author><name sortKey="Wong, S K" uniqKey="Wong S">S.K. Wong</name></author><author><name sortKey="Moore, M J" uniqKey="Moore M">M.J. Moore</name></author><author><name sortKey="Tallarico, A S" uniqKey="Tallarico A">A.S. Tallarico</name></author><author><name sortKey="Olurinde, M" uniqKey="Olurinde M">M. Olurinde</name></author><author><name sortKey="Choe, H" uniqKey="Choe H">H. Choe</name></author><author><name sortKey="Anderson, L J" uniqKey="Anderson L">L.J. Anderson</name></author><author><name sortKey="Bellini, W J" uniqKey="Bellini W">W.J. Bellini</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M. Farzan</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="Hua, R" uniqKey="Hua R">R. Hua</name></author><author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name></author><author><name sortKey="Wang, Y" uniqKey="Wang Y">Y. Wang</name></author><author><name sortKey="Hua, Y" uniqKey="Hua Y">Y. Hua</name></author><author><name sortKey="Tong, G" uniqKey="Tong G">G. Tong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Krokhin, O" uniqKey="Krokhin O">O. Krokhin</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name></author><author><name sortKey="Andonov, A" uniqKey="Andonov A">A. Andonov</name></author><author><name sortKey="Feldmann, H" uniqKey="Feldmann H">H. Feldmann</name></author><author><name sortKey="Flick, R" uniqKey="Flick R">R. Flick</name></author><author><name sortKey="Jones, S" uniqKey="Jones S">S. Jones</name></author><author><name sortKey="Stroeher, U" uniqKey="Stroeher U">U. Stroeher</name></author><author><name sortKey="Bastien, N" uniqKey="Bastien N">N. Bastien</name></author><author><name sortKey="Dasuri, K V" uniqKey="Dasuri K">K.V. Dasuri</name></author><author><name sortKey="Cheng, K" uniqKey="Cheng K">K. Cheng</name></author><author><name sortKey="Simonsen, J N" uniqKey="Simonsen J">J.N. Simonsen</name></author><author><name sortKey="Perreault, H" uniqKey="Perreault H">H. Perreault</name></author><author><name sortKey="Wilkins, J" uniqKey="Wilkins J">J. Wilkins</name></author><author><name sortKey="Ens, W" uniqKey="Ens W">W. Ens</name></author><author><name sortKey="Plummer, F" uniqKey="Plummer F">F. Plummer</name></author><author><name sortKey="Standing, K G" uniqKey="Standing K">K.G. Standing</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhong, X" uniqKey="Zhong X">X. Zhong</name></author><author><name sortKey="Yang, H" uniqKey="Yang H">H. Yang</name></author><author><name sortKey="Guo, Z F" uniqKey="Guo Z">Z.F. Guo</name></author><author><name sortKey="Sin, W Y" uniqKey="Sin W">W.Y. Sin</name></author><author><name sortKey="Chen, W" uniqKey="Chen W">W. Chen</name></author><author><name sortKey="Xu, J" uniqKey="Xu J">J. Xu</name></author><author><name sortKey="Fu, L" uniqKey="Fu L">L. Fu</name></author><author><name sortKey="Wu, J" uniqKey="Wu J">J. Wu</name></author><author><name sortKey="Mak, C K" uniqKey="Mak C">C.K. Mak</name></author><author><name sortKey="Cheng, C S" uniqKey="Cheng C">C.S. Cheng</name></author><author><name sortKey="Yang, Y" uniqKey="Yang Y">Y. Yang</name></author><author><name sortKey="Cao, S" uniqKey="Cao S">S. Cao</name></author><author><name sortKey="Wong, T Y" uniqKey="Wong T">T.Y. Wong</name></author><author><name sortKey="Lai, S T" uniqKey="Lai S">S.T. Lai</name></author><author><name sortKey="Xie, Y" uniqKey="Xie Y">Y. Xie</name></author><author><name sortKey="Guo, Z" uniqKey="Guo Z">Z. Guo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, Z" uniqKey="Chen Z">Z. Chen</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L. Zhang</name></author><author><name sortKey="Qin, C" uniqKey="Qin C">C. Qin</name></author><author><name sortKey="Ba, L" uniqKey="Ba L">L. Ba</name></author><author><name sortKey="Yi, C E" uniqKey="Yi C">C.E. Yi</name></author><author><name sortKey="Zhang, F" uniqKey="Zhang F">F. Zhang</name></author><author><name sortKey="Wei, Q" uniqKey="Wei Q">Q. Wei</name></author><author><name sortKey="He, T" uniqKey="He T">T. He</name></author><author><name sortKey="Yu, W" uniqKey="Yu W">W. Yu</name></author><author><name sortKey="Yu, J" uniqKey="Yu J">J. Yu</name></author><author><name sortKey="Gao, H" uniqKey="Gao H">H. Gao</name></author><author><name sortKey="Tu, X" uniqKey="Tu X">X. Tu</name></author><author><name sortKey="Gettie, A" uniqKey="Gettie A">A. Gettie</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M. Farzan</name></author><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K.Y. Yuen</name></author><author><name sortKey="Ho, D D" uniqKey="Ho D">D.D. Ho</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Biochem Biophys Res Commun</journal-id><journal-id journal-id-type="iso-abbrev">Biochem. Biophys. Res. Commun</journal-id><journal-title-group><journal-title>Biochemical and Biophysical Research Communications</journal-title></journal-title-group><issn pub-type="ppub">0006-291X</issn><issn pub-type="epub">1090-2104</issn><publisher><publisher-name>Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">16630549</article-id><article-id pub-id-type="pmc">7092919</article-id><article-id pub-id-type="publisher-id">S0006-291X(06)00690-5</article-id><article-id pub-id-type="doi">10.1016/j.bbrc.2006.03.152</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>HLA-A<sup>∗</sup>0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Tsao</surname><given-names>Yeou-Ping</given-names></name><xref rid="aff1" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Jian-Yu</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Jan</surname><given-names>Jia-Tsrong</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Leng</surname><given-names>Chih-Hsiang</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Chu</surname><given-names>Chen-Chung</given-names></name><xref rid="aff5" ref-type="aff">e</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Yuh-Cheng</given-names></name><xref rid="aff5" ref-type="aff">e</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Show-Li</given-names></name><email>showlic@ha.mc.ntu.edu.tw</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff6" ref-type="aff">f</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Department of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan</aff><aff id="aff2"><label>b</label>Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Taiwan</aff><aff id="aff3"><label>c</label>Vaccine Research and Development Center, National Health Research Institutes, Taipei 115, Taiwan</aff><aff id="aff4"><label>d</label>Department of Ophthalmology, Mackay Memorial Hospital, Taipei 104, Taiwan</aff><aff id="aff5"><label>e</label>Department of Medical Research, Mackay Memorial Hospital, Taipei 104, Taiwan</aff><aff id="aff6"><label>f</label>Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Fax: +886 2 2391 5293. <email>showlic@ha.mc.ntu.edu.tw</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>3</day><month>4</month><year>2006</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>26</day><month>5</month><year>2006</year></pub-date><pub-date pub-type="epub"><day>3</day><month>4</month><year>2006</year></pub-date><volume>344</volume><issue>1</issue><fpage>63</fpage><lpage>71</lpage><history><date date-type="received"><day>18</day><month>3</month><year>2006</year></date></history><permissions><copyright-statement>Copyright © 2006 Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2006</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><p>The immunogenicity of HLA-A<sup>∗</sup>0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins’ ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (<ext-link ext-link-type="uri" xlink:href="http://thr.cit.nih.gov/molbio/hla_bind/">http://thr.cit.nih.gov/molbio/hla_bind/</ext-link>), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223–231, N227–235, and N317–325 to be the first identified HLA-A<sup>∗</sup>0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A<sup>∗</sup>0201-restricted CTL epitopes of S protein (S978–986, S1203–1211, and S1167–1175), here we found two novel peptides S787–795 and S1042–1050 as S-specific CTL epitopes. Moreover, our identified N317–325 and S1042–1050 CTL epitopes could induce recall responses when IFN-γ stimulation of blood CD8<sup>+</sup> T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.</p></abstract><kwd-group><title>Keywords</title><kwd>Nucleocapsid</kwd><kwd>Spike</kwd><kwd>CTL epitope</kwd><kwd>SARS virus</kwd><kwd>HLA-A<sup>∗</sup>0201</kwd></kwd-group></article-meta></front><body><p id="para.0010">Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus—SARS-associated coronavirus (SARS-CoV). SARS-CoV is an enveloped positive-stranded RNA virus <xref rid="bib1" ref-type="bibr">[1]</xref>, <xref rid="bib2" ref-type="bibr">[2]</xref>. The four structural genes of SARS are S gene, encoding a spike glycoprotein which plays a role in cell–cell fusion; small membrane protein (E), an integral membrane glycoprotein (M), and the N gene encoding a nucleocapsid RNA-binding protein that may be associated with viral M protein <xref rid="bib3" ref-type="bibr">[3]</xref>. Presumably, SARS-CoV infects cells through the S protein, which binds to cell surface receptor-angiotensin-converting enzyme 2 <xref rid="bib4" ref-type="bibr">[4]</xref>. After this initial attachment, the viral E protein fuses with the plasma membrane of the host cell, and a cascade of intracellular events follows, including interaction between the M and N proteins <xref rid="bib4" ref-type="bibr">[4]</xref>. Hence, these four structural proteins are major targets for developing anti-SARS drugs or vaccines. To date many efforts have been made to find a vaccine that can block SARS-CoV infection and eliminate its viral loads.</p><p id="para.0015">Infectious bronchitis virus (IBV), a prototype of the Coronaviridae family, causes a highly contagious respiratory disease of chickens. IBV-specific CTLs are critical to play a role in the elimination of virus particles during acute infection and subsequent control of the infection in chickens <xref rid="bib5" ref-type="bibr">[5]</xref>, <xref rid="bib6" ref-type="bibr">[6]</xref>. Therefore, CTL may determine the role of immunity in controlling viral pathogenesis. In this project, for generating SARS-CoV CTL response, we chose to analyze the two structural genes-S and N for defining HLA-A<sup>∗</sup>0201-restricted CTL epitope for the future vaccination. Human CTLs are specific for peptides presented in the context of major histocompatibility complex (MHC) molecules. Prior to presentation, peptides are generated in the cytosol by limited proteolytic fragmentation of all available antigens, translocated to the endoplasmic reticulum, specifically sampled by MHC molecules, and exported to the cell surface, where they await CTL scrutiny. Subunit vaccines that contain small synthetic peptides corresponding to minimal cytotoxic T lymphocyte (CTL) epitopes have been shown to be highly effective for the induction of strong, protective CTL-mediated immunity against infectious virus and tumor growth in murine models <xref rid="bib7" ref-type="bibr">[7]</xref>. Hence, in this study, we mapped the HLA-A<sup>∗</sup>0201 CTL epitopes of SARS-CoV N and S proteins. We used online database analysis to predict HLA binding peptides of SARS-CoV N and S proteins and validated by T2-cell binding assay <xref rid="bib8" ref-type="bibr">[8]</xref>. Our results identified three N- and two S-specific HLA-A<sup>∗</sup>0201 peptides. Moreover, our identified N317–325 and S1042–1050 CTL epitopes could induce recall responses when IFN-γ stimulation of blood CD8<sup>+</sup> T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with its cognate antigen.</p><sec id="section.0010"><title>Materials and methods</title><p id="para.0020"><italic>Peptide synthesis.</italic> To determine potential vaccine candidates, ten each potential 9-amino acid peptides of SARS-CoV N and S proteins were synthesized. In addition, the HLA-A2.1-binding peptide YMDGTMSQV (tyrosinase 369–377) and the HLA-A1-binding peptide EADPTGHSY (MAGE-1 161–169) <xref rid="bib9" ref-type="bibr">[9]</xref> were tested positive and negative control, respectively. These peptides were synthesized by solid-phase strategies on an automated peptide synthesizer (Abimed AMS 422) using Fmoc chemistry. Peptides were analyzed by reverse-phase high-performance liquid chromatography (HPLC) and dissolved in dimethyl sulfoxide (DMSO) at 1–5 mg/ml, liquated, and stored at −70 °C.</p><p id="para.0025"><italic>T2-cell stabilization assay: stabilization of HLA-A2.1 on the surface of T2-cells by synthetic peptides.</italic> To determine whether synthetic peptides could bind to HLA-A<sup>∗</sup>0201 molecules, peptide-induced HLA-A<sup>∗</sup>0201 up-regulation on T2-cells was examined according to a protocol described previously <xref rid="bib8" ref-type="bibr">[8]</xref>. To measure the relative amounts of HLA-A2.1-peptide complexes formed, 2 × 10<sup>6</sup> cells were incubated with 100 μg of each N or S protein peptide for 3.5 h at 26 °C in HBSS supplemented with 5% FBS, 5 μg/ml human β 2-microglobulin (Sigma), and 5 μg/ml brefeldin A. The various titrations of peptide for T2-cell binding assay were tested, and 100 μg of peptide was the approximate concentration for this assay (data not shown). The cells were then washed and incubated for an additional 2 h at 37 °C to allow the remaining peptide-free MHC molecules to denature, followed by indirect immunofluorescence staining for HLA-A2.1 expression with BB7.2 Ab and goat anti-mouse IgG (F(ab’)2) FITC (Jackson ImmunoResearch, West Grove, PA), being fixed with 1% paraformaldehyde, and being analyzed by flow cytometry. Results were calculated as a percentage of HLA-A2.1 expression using the formula: (fluorescence intensity of experimental peptide binding to HLA-A2.1 − mock peptide binding to HLA-A2.1) × 100%.</p><p id="para.0030"><italic>Production of recombinant N protein.</italic> The recombinant N protein was expressed from pRSETA vector (Invitrogen, Carlsbad, CA, USA) in <italic>Escherichia coli</italic> system BL21(DE3)Gold (Stratagene, Cedar Creek, TX, USA) by DNA recombinant technology and purified by Ni–NTA resin as reported by Liu et al. <xref rid="bib10" ref-type="bibr">[10]</xref>.</p><p id="para.0035"><italic>Animals.</italic> HLA-A2.1 transgenic mice were purchased and imported from the Jackson Laboratory, and maintained in our Institute under specific pathogen-free conditions. The transgenic mice with C57BL/6 background that express a MHC-I molecule of human HLA-A2.1 have been described previously <xref rid="bib11" ref-type="bibr">[11]</xref>. These transgenic mice received each N- or S-specific peptide (100 μg) or N protein (50 μg) with 30 μg CpG oligodeoxynucleotide (CpG ODN 1826) via intramuscular injection three times at one week intervals. One week after the last immunization, we mapped the CTL epitopes of N and S proteins by analyzing antigen-specific CD8<sup>+</sup> interferon-γ<sup>+</sup> (IFN-γ<sup>+</sup>) double-positive cells in HLA-A2.1 transgenic mice with flow cytometry <xref rid="bib12" ref-type="bibr">[12]</xref>, <xref rid="bib13" ref-type="bibr">[13]</xref>.</p><p id="para.0040"><italic>Intracytoplasmic cytokine staining and flow cytometry analysis.</italic> Splenocytes (2 × 10<sup>6</sup>) from peptide vaccinated HLA-A2.1 transgenic mice or controls were incubated for 12 h with a 30 μg stimulator (each N or S synthetic peptide) for the detection of N or S peptide-specific CD8<sup>+</sup> T-cell precursors. Golgistop (PharMingen, San Diego, CA) was added into each well at 6 h before the cells were harvested. Cells were then washed twice in FACScan buffer and stained with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD8b.2 (Ly-3.2) monoclonal antibody (PharMingen). Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer’s instructions (PharMingen). Phycoerythrin-conjugated rat anti-mouse IFN-γ monoclonal antibody was purchased from PharMingen. FACS analysis was performed on a Becton–Dickinson FACScan with CELLQuest software (Becton–Dickinson Immunocytometry System, Mountain View, CA).</p><p id="para.0045"><italic>HLA-A</italic><sup>∗</sup><italic>0201 typing.</italic> Blood samples were collected in acid citrate dextrose tubes and transported within 24 h to the Immunohematology Reference Laboratory, Mackay Memorial Hospital (Taipei, Taiwan). All samples were tested for HLA-A, B, C serology (standard microlymphocytotoxicity technique) using Terasaki Chinese HLA-ABC 72-Well Trays (lots 2, 3, 3A, 3B) <xref rid="bib14" ref-type="bibr">[14]</xref>.</p><p id="para.0050"><italic>In vitro vaccination and IFN-γ assay.</italic> Samples were obtained with informed consent from Tri-Service General Hospital and Mackay Memorial Hospital, Taipei, Taiwan, and the study was approved by the institutional Human Investigation Review Committee. Human PBMCs were isolated, respectively from 5 normal and 5 full recovery patients of severe SARS-infection with HLA-A<sup>∗</sup>0201 type by separation on Ficoll–Hypaque density gradients and re-suspended in T-cell medium <xref rid="bib15" ref-type="bibr">[15]</xref>. These five recovery patients including one laboratory worker and four hospital nurses contracted SARS-CoV during the epidemic period on 2003 <xref rid="bib16" ref-type="bibr">[16]</xref>. They were afflicted with SARS disease and hospitalized until full recovery. All of them were identified virus RNA in respiratory secretions. Antibodies against SARS-CoV were detected by SARS-specific IgG capture ELISA and end-point dilution Ab titers. The Ab titers were ≧1:16 and were observed in all recovered patients. No Ab to SARS-CoV was detected in any healthy donors. All patients’ bloods were harvested over one year post-infection.</p><p id="para.0055">The isolated peripheral blood mononuclear cells were incubated on plastic dishes for 2 h. The non-adherent lymphocytes were aspirated, the adherent fraction was cultured in medium containing 1% polled human AB serum, recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (1000 IU/ml), and recombinant human interleukin 4 (IL-4) (1000 IU/ml) (R and D Systems, Minneapolis, Minn.). One hundred micrograms of recombinant N protein or the combination of multiple S peptides was incubated with the adherent cells at 37 °C. Recombinant human interleukin 2 (IL-2) (10 IU/ml), recombinant human interleukin 7 (IL-7) (10 IU/ml), recombinant human interleukin 1-beta (IL-1β) (10 ng/ml), and recombinant human tumor necrosis factor-alpha (TNF-α) (10 ng/ml) were added every second days into antigen-vaccinated adherent cells. Three days after the initial addition of antigens to adherent cells, autologous blood lymphocytes (non-adherent cells) (2 × 10<sup>6</sup>/well) were co-incubated with adherent cells, on the second day recombinant human interleukin 2 (IL-2) (10 IU/ml) was added. Cultures were fed with medium containing penicillin, streptomycin, IL-2, and IL-7. After 7 to 15 days of culture, the CD8<sup>+</sup> T-cells were purified by labeling blood lymphocytes with magnetic microbeads coated with anti-CD8<sup>+</sup> antibody in accordance with the manufacturer’s recommendations (Miltenyi Biotech GmbH) and separated on an Automacs device (Miltenyi Biotech GmbH) using the series procedures <xref rid="bib17" ref-type="bibr">[17]</xref>. The isolated CD8<sup>+</sup> T-cells were then stimulated with each indicated peptide, and the next day, the supernatants were collected and analyzed for IFN-γ (polyclonal Ab) with an ELISA kit (Quantikine, R&D systems). The series concentrations of standard protein (0, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 pg/ml) were parallely measured for the reference, accordingly the concentrations of IFN-γ in each test were calculated.</p></sec><sec id="section.0015"><title>Results</title><sec id="section.0020"><title>Define HLA-A<sup>∗</sup>0201-restricted epitopes of N and S proteins of SARS-CoV by T2-cell binding assay</title><p id="para.0060">We screened the amino acid sequences of N and S proteins of SARS-CoV and used HLA peptide binding predictions offered by the National Institute of Health (NIH) (<ext-link ext-link-type="uri" xlink:href="http://thr.cit.nih.gov/molbio/hla-bind/">http://thr.cit.nih.gov/molbio/hla-bind/</ext-link>) to determine the potential CTL epitopes by estimating dissociation half-life from MHC class I molecules. Ten each potential N- and S-specific HLA-A2-binding peptides along with the positive and negative control peptides were then synthesized (<xref rid="app1" ref-type="sec">Supplement Table 1</xref>) and subjected to a T2-cell binding assay for validating the binding affinity of HLA-A2.1. The tyrosine-derived peptide YMDGTMSQV served as a positive control <xref rid="bib9" ref-type="bibr">[9]</xref>, and the HLA-A1-binding peptide EADPTGHSY (MAGE-1) served as a negative control. Online database analysis and the T2-cell binding affinity assay revealed that three N peptides (N223–231, N227–235, and N317–325) (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A) and two S peptides (S787–795 and S1042–1050) (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B) exhibited strong binding to HLA-A2.1 molecules.<fig id="fig1"><label>Fig. 1</label><caption><p>Stabilization of HLA-A<sup>∗</sup>0201 molecules on the surface of T2-cells by computer predicted peptides of SARS-CoV N and S protein. The HLA-A2.1 expression was determined by FACS staining with the BB7.2 mAb, and the median fluorescence intensity calculated, respectively. (A) T2-cells were incubated with N peptides and (B) T2-cells were incubated with S peptides. Three independent experiments were involved in each column and the summarized data shown in the histogram. YMDG, the HLA-A2.1 tyrosinase-derived peptide YMDGTMSQV served as a positive control. EADP, the HLA-A1-binding peptide EADPTGHSY (MAGE-1) served as a negative control.</p></caption><graphic xlink:href="gr1"></graphic></fig></p></sec><sec id="section.0025"><title>SARS-CoV N and CTL epitopes mapped by vaccinating HLA-A2.1 transgenic mice with recombinant N protein</title><p id="para.0065">Transgenic mice have been widely used as preclinical disease models and can provide information relevant to clinical trial design. Transgenic mice with a C57BL/6 background expressing class I MHC molecules of human HLA-A2.1 have been described previously <xref rid="bib11" ref-type="bibr">[11]</xref>. Using these mice, we mapped HLA-A2.1 CTL epitopes by vaccinating HLA-A2.1 transgenic mice with 50 μg recombinant N protein three times at one week intervals. One week after the last vaccination, splenocytes were harvested and stimulated with each of the T2-cell binding peptides or non-binding peptides separately, after which CTLs were assayed by CD8<sup>+</sup> IFN-γ<sup>+</sup> double staining with flow cytometry <xref rid="bib18" ref-type="bibr">[18]</xref>. As shown in <xref rid="fig2" ref-type="fig">Fig. 2</xref>A, peptides N223–231, N227–235, and N317–325 stimulation resulting 38, 75, and 121 CD8<sup>+</sup> IFN-γ<sup>+</sup> T-cells were detected per 10<sup>5</sup> splenocytes derived from SARS-CoV N protein vaccination, it was an approximate 3-, 4-, and 8-fold increase in N223–231, N227–235, and N317–325 peptide stimulation compared to N407–415 (non T2-cell binding peptide), respectively (<xref rid="fig2" ref-type="fig">Fig. 2</xref>B). As to IFN-γ production from non-CD8<sup>+</sup> cells in <xref rid="fig2" ref-type="fig">Fig. 2</xref>A, it suggests that CD4 or natural killer cells may involve in and that is worth for further investigation. To sum, peptides N223–231, N227–237, and N317–325 could act as stimulators to induce a SARS-CoV N-specific CTL response.<fig id="fig2"><label>Fig. 2</label><caption><p>HLA-A<sup>∗</sup>0201-restricted epitopes of SARS N protein defined by vaccination of HLA-A2.1 transgenic mice with recombinant N protein. HLA-A2.1 transgenic mice (four to six weeks old) were vaccinated three times by the recombinant N protein at one week intervals. One week after the last immunization, splenocytes from the vaccinated HLA-A2.1 transgenic mice were harvested and stimulated with each N peptide, then intracellular cytokine staining with flow cytometry was performed to determine the number of CD8<sup>+</sup> IFN-γ<sup>+</sup> double-positive cells. (A) Splenocytes from vaccinated mice were stimulated in vitro with peptides and stained with CD8<sup>+</sup> and IFN-γ<sup>+</sup> antibodies. The number of CD8<sup>+</sup> and IFN-γ<sup>+</sup> double-positive cells was analyzed by flow cytometry and shown in the upper corners of each panel. The result of one representative assay from three similar independent experiments is shown. (B) Summarizes the three independent experiments. The data represent the means and standard error of three experiments. <italic>Y</italic>-axis denotes fold increase in antigen-specific splenocytes = (vaccinated splenocytes stimulated with the test peptide)/(vaccinated splenocytes stimulated with control peptide) × 100%.</p></caption><graphic xlink:href="gr2"></graphic></fig></p></sec><sec id="section.0030"><title>Identification of SARS-CoV N and S CTL epitopes from HLA-A2.1 transgenic mice vaccinated with T2-cell binding peptides plus CpG ODN 1826</title><p id="para.0070">Immunization experiments were conducted with the three T2-cell binding peptides of N protein and two S peptides as well as each non-T2-cell binding peptide-N407–415 and S151–159 (<xref rid="fig1" ref-type="fig">Figs. 1</xref>A and B), to test its capacity to induce peptide-specific CTL immunity in vivo. CpG phosphorothioate oligodeoxynucleotide 1826 (CpG ODN 1826) was used as an adjuvant <xref rid="bib18" ref-type="bibr">[18]</xref>. Transgenic mice received each peptide with CpG ODN 1826 via intramuscular injection three times at one week intervals. One week after third immunization, we measured peptide-specific CTL activity by analyzing antigen-specific CD8<sup>+</sup>IFN-γ<sup>+</sup> double-positive cells in HLA-A2.1 transgenic mice with flow cytometry. As shown in <xref rid="fig3" ref-type="fig">Figs. 3</xref>A and B, CTL activity resulting from vaccination with peptides N223–231, N227–235, and N317–325 was significantly increased by about 4-, 4-, and 5-fold, respectively, compared to non-T2-cell binding peptide-N407–415. The cutoff for significant immune response in this study was chosen about fourfold differences between stimulator peptide and irrelevant peptide-inducing CTLs. Likewise, to determine S CTL epitopes, CTL activity resulting from vaccination with peptides S787–795 and S1042–1050 was increased by about 4- and 5-fold, respectively (<xref rid="app1" ref-type="sec">Supplement Fig. 1</xref>), indicating that peptides S787–795 and S1042–1050 were immunogenic CTL epitopes of SARS-CoV S protein.<fig id="fig3"><label>Fig. 3</label><caption><p>Identification of HLA-A<sup>∗</sup>0201-restricted epitopes of SARS N protein by vaccination of HLA-A2.1 transgenic mice with T2 binding peptide plus CpG ODN 1826. Four- to six-week-old HLA-A2.1 transgenic mice were vaccinated three times with T2-cells binding peptide plus CpG ODN 1826 at one week intervals. One week after the last vaccination, splenocytes were harvested and stimulated with each indicated peptide; then intracellular cytokine staining with flow cytometry was performed as described in <xref rid="fig2" ref-type="fig">Fig. 2</xref>. (A) Splenocytes from vaccinated mice were stimulated in vitro with N peptide and stained with CD8<sup>+</sup> and IFN-γ<sup>+</sup> antibodies. The number of CD8<sup>+</sup> and IFN-γ<sup>+</sup> double-positive cells was analyzed by flow cytometry and shown in the upper corners of each panel. The result of one representative assay from three similar independent experiments is shown. (B) Summarizes the three independent experiments. The data represent the means and standard error of three experiments. <italic>Y</italic>-axis denotes fold increase in antigen-specific splenocytes = (vaccinated splenocytes stimulated with the test peptide)/(vaccinated splenocytes stimulated with control peptide) × 100%.</p></caption><graphic xlink:href="gr3"></graphic></fig></p></sec><sec id="section.0035"><title>HLA-A<sup>∗</sup>0201 T-cell epitopes of SARS-CoV N and S protein stimulate specific immune response in convalescent SARS patients and healthy donors</title><p id="para.0075">Recently several groups measured vaccine efficacy by in vitro vaccinating PBMCs from cancer patients with peptides <xref rid="bib7" ref-type="bibr">[7]</xref>, tumor cells <xref rid="bib19" ref-type="bibr">[19]</xref>, soluble protein <xref rid="bib20" ref-type="bibr">[20]</xref>, virus like particles <xref rid="bib15" ref-type="bibr">[15]</xref>, and recombinant adenoviruses <xref rid="bib21" ref-type="bibr">[21]</xref>. They all showed that in vitro vaccination with these agents could trigger human CTLs against tumor antigens. Moreover, the recent report demonstrated that in vitro vaccination of healthy donors with human papillomavirus type 16 E7 protein can generate E7-specific CTL clones <xref rid="bib22" ref-type="bibr">[22]</xref>. It indicates that CTL clones can be generated by in vitro vaccinating PBMCs from either patients or normal healthy. For avoiding radioactive risk, we used ELISA to measure IFN-γ releasing from purified CD8<sup>+</sup> T-cells as a CTL assay instead of chromium release assay and ELISPOT assay. We had performed ELISPOT and ELISA for IFN-γ production of purified CD8<sup>+</sup> T-cells in parallel, the results of these two assays were coherent (data not shown). Therefore in this study, we examined CTL activity by IFN-γ releasing from purified CD8<sup>+</sup> T-cells with ELISA kit. The lymphocytes were obtained from five HLA-A<sup>∗</sup>0201 donors who had fully recovered from SARS-CoV infection and five HLA-A<sup>∗</sup>0201 healthy donors with no history of SARS-CoV infection. The adherent and non-adherent cells were isolated and divided from human bloods by separation on Ficoll–Hypaque density gradients as described in Materials and methods. Recombinant N protein or the combination peptides of S protein were introduced into isolated adherent cells. The vaccinated adherent cells were then co-cultured with autologous lymphocytes (non-adherent cells). Two weeks after co-incubation, CD8<sup>+</sup> T-cells were isolated. The purification of CD8<sup>+</sup> T-cells was analyzed by flow cytometry, and the results showed no CD4<sup>+</sup> T-cells and NK cells existing (data not shown). The isolated CD8<sup>+</sup> T-cells were then stimulated with each peptide, and IFN-γ<sup>+</sup> production was assayed by ELISA. As shown in <xref rid="fig4" ref-type="fig">Fig. 4</xref>A, stimulation with peptides N223–231 or N227–235 or N317–325 produced high amounts of IFN-γ<sup>+</sup> both in normal healthy and fully recovered SARS-CoV infected patients, but a low response to irrelevant peptide. The positive control was T-cell response to HLA-A2-restricted influenza virus matrix peptide stimulation <xref rid="bib23" ref-type="bibr">[23]</xref>. There was no significant T-cell response in non-HLA-A<sup>∗</sup>0201 of healthy donors and SARS-CoV infected patients (data not shown). These data, derived from an average five individuals in each group indicated that human antigen presenting cells can process N protein, therefore present MHC I molecules complexed with N peptide to T-cells. Therefore peptides N223–231, N227–235, and N317–325 are endogenous CTL epitopes of N protein. Likewise, in vitro vaccination data showed peptides S787–795 and S1042–1050 as S-specific CTL epitopes (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B). Furthermore, to look into the recall response of CTL peptides, the IFN-γ production from blood CD8<sup>+</sup> T-cells in response to N317–327 and S1042–1050 CTL epitope stimulation revealed significant difference between normal healthy donors and SARS-recovery patients after those PBMCs were in vitro vaccinating with their cognate antigen, indicating that these two CTL peptides in recovery patients can induce higher immune response than normal healthy (<xref rid="fig4" ref-type="fig">Figs. 4</xref>A and B). However, N223–231, N227–235, and S787–795 CTL epitope stimulation showed no significant difference between normal donors and SARS-recovered patients, indicating that no-recall response of these three peptides exists in blood lymphocytes of fully convalescent SARS-CoV infected patients.<fig id="fig4"><label>Fig. 4</label><caption><p>Identification of HLA-A<sup>∗</sup>0201-restricted CTL epitope of SARS N and S proteins via in vitro vaccination of human blood lymphocytes with recombinant N protein (A) and the mixture of several S peptides (B). Human peripheral blood lymphocytes were co-cultured with autologous adherent cells that were incubated with recombinant N protein or S peptides as described in Materials and methods. The isolated CD8<sup>+</sup> T-cells were stimulated with each indicated peptide. The IFN-γ production of CD8<sup>+</sup> was determined by ELISA. Gray column, from normal healthy donors. Black column, from SARS recovered patients. The data represent the means and standard error of tested samples after subtraction of background responses. <italic>Y</italic>-axis denotes the concentration of IFN-γ production. The positive control (INF) was validated by stimulation with HLA-A<sup>∗</sup>0201-restricted influenza virus matrix peptide (GILGFVFTL). * Indicates statistically significant difference by <italic>t</italic> test (<italic>p</italic> < 0.05).</p></caption><graphic xlink:href="gr4"></graphic></fig></p></sec></sec><sec id="section.0040"><title>Discussion</title><p id="para.0080">In this study, we were the first to identify N peptides 223–231, 227–235, and 317–325 as HLA-A<sup>∗</sup>0201-restricted CTL epitopes of SARS-CoV N protein; and two novel S CTL epitopes S787–795 and S1042–1050 in addition to three S epitopes (978–986, 1203–1211, and 1167–1175) already known <xref rid="bib24" ref-type="bibr">[24]</xref>, <xref rid="bib25" ref-type="bibr">[25]</xref> by immunizing HLA-A2.1 transgenic mice and in vitro vaccinating HLA-A<sup>∗</sup>0201 positive human PBMCs to measure their immunogenicity. During SARS-CoV infection, N protein is reported to be highly immunogenic and large quantities have been found in serology assays at the early stage of SARS-CoV infected patients <xref rid="bib26" ref-type="bibr">[26]</xref>, <xref rid="bib27" ref-type="bibr">[27]</xref>. Similarly, the N antigen also has been found to be highly immunogenic in elk CoV <xref rid="bib28" ref-type="bibr">[28]</xref> and IBV <xref rid="bib29" ref-type="bibr">[29]</xref>. However until now, there has been no report concerning any N-specific CTL epitopes to elicit cellular immune response in humans. But there are several published papers for SARS-CoV N protein vaccine development in experimental animals. For examples, N vaccines can efficiently generate N-specific humoral and T-cell-mediated immune responses in vaccinated mice or monkey <xref rid="bib30" ref-type="bibr">[30]</xref>, <xref rid="bib31" ref-type="bibr">[31]</xref>. In this study, we are the first to identify three N-specific HLA-A<sup>∗</sup>0201 CTL epitopes N223–231, N227–235, and N317–325 that can stimulate IFN-γ release from CD8<sup>+</sup> T-cells via in vitro vaccination of human blood lymphocytes from healthy donors or SARS-CoV fully recovered patients. Therefore, these three T-cell epitope peptides are good candidates for at least the partial constitution of an anti-SARS-CoV vaccine.</p><p id="para.0085">The S of SARS-CoV, a 1255-amino acid type I membrane glycoprotein <xref rid="bib32" ref-type="bibr">[32]</xref>, is the prominent protein present in the viral membrane and presents as the typical spike structure found on all coronaviruses <xref rid="bib33" ref-type="bibr">[33]</xref>. S glycoprotein domain structure has been deduced from sequence analysis <xref rid="bib32" ref-type="bibr">[32]</xref>. The S glycoprotein consists of a leader (amino acids 1 to 14), an ectodomain represented by amino acids 15 to 1190, a membrane spanning domain (amino acids 1191 to 1227), and a short intracellular tail (amino acids 1227 to 1255) <xref rid="bib32" ref-type="bibr">[32]</xref>. Previously, the three S-specific HLA-A<sup>∗</sup>0201-restricted CTL epitopes were reported <xref rid="bib24" ref-type="bibr">[24]</xref>, <xref rid="bib25" ref-type="bibr">[25]</xref>. In this study, we found two novel S-specific HLA-A<sup>∗</sup>0201 CTL epitopes S787–795 and S1042–1050. Wang et al. <xref rid="bib24" ref-type="bibr">[24]</xref> used SYFPEITHI and EpitopePredict <xref rid="bib24" ref-type="bibr">[24]</xref> to predict S-specific HLA-A<sup>∗</sup>0201 binding affinity epitopes and identified peptides S978–986 and S1203–1211 as S-specific HLA-A<sup>∗</sup>0201-restricted CTL epitopes. In our study, we used the NIH database to predict potential HLA-A<sup>∗</sup>0201 binding peptides. The identification of S787–795 and S1042–1050 as CTL epitopes of S protein was validated by a T2-cell binding assay and the immunogenicity was measured by HLA-A2.1 transgenic mice and in vitro vaccination of human PBMCs. As to identification of CTL epitopes of S protein, unlike N protein, there was not available the entire S protein for vaccination in this study, but induction of CTL activity via in vitro vaccination of human blood lymphocytes with S787–795 and S1042–1050 found in this study had a similar stimulating effect as S978–986 and S1203–1211 identified by Wang et al. <xref rid="bib24" ref-type="bibr">[24]</xref> (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B). Hence, S787–795 and S1042–1050 can be regarded as two novel S-specific HLA-A<sup>∗</sup>0201 CTL epitopes.</p><p id="para.0090">Recently, angiotensin-converting enzyme 2 (ACE2) was found to be a functional receptor for SARS-CoV S protein <xref rid="bib4" ref-type="bibr">[4]</xref>. Subsequently, the ACE2 receptor-binding domain was located between amino acid residues 303 and 537 of the S <xref rid="bib34" ref-type="bibr">[34]</xref>, <xref rid="bib35" ref-type="bibr">[35]</xref>, <xref rid="bib36" ref-type="bibr">[36]</xref>. Therefore if including the results of the present study, five of the known S-specific CTL epitopes (S787–785, S1042–1050, S978–986, S1203–1211, and S1167–1175) seem not to be involved in ACE2 receptor binding of host <xref rid="bib24" ref-type="bibr">[24]</xref>, <xref rid="bib25" ref-type="bibr">[25]</xref>, <xref rid="bib35" ref-type="bibr">[35]</xref>, <xref rid="bib36" ref-type="bibr">[36]</xref>. Hence, the binding domain (amino acid 303 to 537) of S protein to ACE2 receptor is not strong CTL activity in HLA-A<sup>∗</sup>0201 humans. On the other hand, B-cell epitopes of spike protein have been found. Among those, the epitope covering 789–799 amino acids of S protein is reported to be B-cell antigenic epitopes by the recognition of monoclonal antibodies <xref rid="bib37" ref-type="bibr">[37]</xref>. The other recent report has also revealed that the amino acids between 787 and 809 are an immunodominant site which the human humoral immune system recognizes and interacts with. This short peptide fragment (787–809) is located in the loop region between the predicted N and M helices of the spike protein and is in close proximity to a glycosylation site identified at glutamine 783 <xref rid="bib38" ref-type="bibr">[38]</xref>, suggesting that this fragment is on the surface of the spike protein. Our data combining with these above results, reveals that the T-cell epitope S787–795 identified in this study may elicit both strong humoral and cellular immunity <xref rid="bib39" ref-type="bibr">[39]</xref>, and may be a more valuable candidate for vaccine development. In this study, CD8<sup>+</sup> T-cells from normal healthy donors could induce T-cell response to N- or S-peptide stimulation after whose blood lymphocytes were in vitro vaccinated with its cognate antigen (<xref rid="fig4" ref-type="fig">Figs. 4</xref>A and B). It indicates that either N or S antigens may stimulate normal human blood T-cells to become effective CTLs in vitro. The recent studies have similarly reported that heat-inactivated SARS-CoV can elicit CTL response <xref rid="bib40" ref-type="bibr">[40]</xref> and human papillomavirus type 16 E7-specific CTL clones can be generated from in vitro vaccinating normal healthy blood lymphocytes <xref rid="bib22" ref-type="bibr">[22]</xref>. This would suggest that mature dendritic cells have the capability to activate SARS-CoV N- and S-specific CTLs from naı¨ve precursor in vitro.</p><p id="para.0095">In this study, N peptides N223–231, N227–235, N317–325 as well as S peptides S787–795 and S1042–1050 were identified as N- and S-specific HLA-A<sup>∗</sup>0201 CTL epitopes, respectively. Among them, CTL peptides, N317–325 and S1042–1050 could induce recall response, but N223–231, N227–235, and S787–795 CTL epitopes did not (<xref rid="fig4" ref-type="fig">Fig. 4</xref>). Since the IFN-γ production by N317–325 and S1042–1050 CTL epitopes stimulation revealed significant difference between normal healthy donors and SARS-recovery patients after those PBMCs were in vitro vaccinating with cognate antigen, indicating that these two CTL peptides can trigger recall responses (<xref rid="fig4" ref-type="fig">Fig. 4</xref>). However, the IFN-γ secretion by N223–231, N227–235, and S787–795 CTL epitopes stimulation showed no significant difference between normal donors and SARS-recovered patients, indicating that no-recall response of these three peptides exists in blood lymphocytes of fully convalescent SARS-CoV infected patients. Previous report identified two S-specific HLA-A<sup>∗</sup>0201 CTL epitopes S978–986 and S1203–1211 <xref rid="bib24" ref-type="bibr">[24]</xref>, T-cell specific immune response to these two epitopes (S978–986 and S1203–1211) was only observed in HLA-A<sup>∗</sup>0201 and SARS-CoV infected patients but not in healthy donors. But when we tested these two peptides (S978–986 and S1203–1211) along with our finding CTL epitopes, the results showed that S978–986 and S1203–1211 elicited no difference in immune response between healthy donors and recovered patients (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B). The different results of S978–986 and S1203–1211 in their and our assays are in assay timing <xref rid="bib24" ref-type="bibr">[24]</xref>. In their study, T-cell immunity was analyzed one month after SARS-CoV infection; but the recovered patients in our study were tested from at least one year after virus infection. This would suggest that the memory cells to S978–986 and S1203–1211 existing in the blood of SARS-CoV recovered patient may not be long-lived. It was confirmed in their continuing report that S978–986 and S1203–1211 do not elicit Ag-specific recall response when analyzed CTL responses of PBMC to inactivated SARS-CoV in SARS-recovered patients over one year after recovery <xref rid="bib40" ref-type="bibr">[40]</xref>. In sum, the memory T-cells to N317–325 and S1042–1050 seem long-lived; hence, these two peptides become more valuable candidates for future vaccination and diagnosis marker of SARS prevention and infection. Due to the decline in the SARS epidemic, no acute SARS patients are available to illustrate the balance between the pathogenesis and immune reaction of SARS. Our results would provide a new insight in to the development of immune response of SARS-CoV infection and the approaches of therapeutic vaccine in SARS.</p></sec></body><back><ref-list id="bibliography.0010"><title>References</title><ref id="bib1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ksiazek</surname><given-names>G.</given-names></name><name><surname>Endman</surname><given-names>D.</given-names></name><name><surname>Goldsmith</surname><given-names>C.S.</given-names></name><name><surname>Zaki</surname><given-names>S.R.</given-names></name><name><surname>Peret</surname><given-names>T.</given-names></name><name><surname>Emery</surname><given-names>S.</given-names></name><name><surname>Tong</surname><given-names>S.</given-names></name><name><surname>Urbani</surname><given-names>C.</given-names></name><name><surname>Comer</surname><given-names>J.A.</given-names></name><name><surname>Lim</surname><given-names>W.</given-names></name><name><surname>Rollin</surname><given-names>P.E.</given-names></name><name><surname>Dowell</surname><given-names>S.F.</given-names></name><name><surname>Ling</surname><given-names>A.E.</given-names></name><name><surname>Humphrey</surname><given-names>C.D.</given-names></name><name><surname>Shieh</surname><given-names>W.J.</given-names></name><name><surname>Guarner</surname><given-names>J.</given-names></name><name><surname>Paddock</surname><given-names>C.D.</given-names></name><name><surname>Rota</surname><given-names>P.</given-names></name><name><surname>Fields</surname><given-names>B.</given-names></name><name><surname>DeRisi</surname><given-names>J.</given-names></name><name><surname>Yang</surname><given-names>J.Y.</given-names></name><name><surname>Cox</surname><given-names>N.</given-names></name><name><surname>Hughes</surname><given-names>J.M.</given-names></name><name><surname>LeDuc</surname><given-names>J.W.</given-names></name><name><surname>Bellini</surname><given-names>W.J.</given-names></name><name><surname>Anderson</surname><given-names>L.J.</given-names></name><name><surname>Group</surname><given-names>S.W.</given-names></name></person-group><article-title>A novel coronavirus associated with severe acute respiratory syndrome</article-title><source>N. Engl. J. Med.</source><volume>348</volume><year>2003</year><fpage>1953</fpage><lpage>1966</lpage><pub-id pub-id-type="pmid">12690092</pub-id></element-citation></ref><ref id="bib2"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Poutanen</surname><given-names>M.</given-names></name><name><surname>Low</surname><given-names>E.</given-names></name><name><surname>Henry</surname><given-names>B.</given-names></name><name><surname>Finkelstein</surname><given-names>S.</given-names></name><name><surname>Rose</surname><given-names>D.</given-names></name><name><surname>Green</surname><given-names>K.</given-names></name><name><surname>Tellier</surname><given-names>R.</given-names></name><name><surname>Draker</surname><given-names>R.</given-names></name><name><surname>Adachi</surname><given-names>D.</given-names></name><name><surname>Ayers</surname><given-names>M.</given-names></name><name><surname>Chan</surname><given-names>A.K.</given-names></name><name><surname>Skowronski</surname><given-names>D.M.</given-names></name><name><surname>Salit</surname><given-names>I.</given-names></name><name><surname>Simor</surname><given-names>A.E.</given-names></name><name><surname>Slutsky</surname><given-names>A.S.</given-names></name><name><surname>Doyle</surname><given-names>P.W.</given-names></name><name><surname>Krajden</surname><given-names>M.</given-names></name><name><surname>Petric</surname><given-names>M.</given-names></name><name><surname>Brunham</surname><given-names>R.C.</given-names></name><name><surname>McGeer</surname><given-names>A.J.</given-names></name><name><surname>C. National Microbiology Laboratory</surname></name><name><surname>C. S. A. R. S. S. Team</surname></name></person-group><article-title>Identification of severe acute respiratory syndrome in Canada</article-title><source>N. Engl. J. Med.</source><volume>56</volume><year>2003</year><fpage>582</fpage><lpage>588</lpage></element-citation></ref><ref id="bib3"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><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>L.A.</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></person-group><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><year>2003</year><fpage>1779</fpage><lpage>1785</lpage><pub-id pub-id-type="pmid">12781537</pub-id></element-citation></ref><ref id="bib4"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><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><name><surname>Berne</surname><given-names>M.A.</given-names></name><name><surname>Somasundaran</surname><given-names>M.</given-names></name><name><surname>Sullivan</surname><given-names>J.L.</given-names></name><name><surname>Luzuriaga</surname><given-names>K.</given-names></name><name><surname>Greenough</surname><given-names>T.C.</given-names></name><name><surname>Choe</surname><given-names>H.</given-names></name><name><surname>Farzan</surname><given-names>M.</given-names></name></person-group><article-title>Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus</article-title><source>Nature</source><volume>426</volume><year>2003</year><fpage>450</fpage><lpage>454</lpage><pub-id pub-id-type="pmid">14647384</pub-id></element-citation></ref><ref id="bib5"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pei</surname><given-names>J.</given-names></name><name><surname>Briles</surname><given-names>W.E.</given-names></name><name><surname>Collisson</surname><given-names>E.W.</given-names></name></person-group><article-title>Memory T cells protect chicks from acute infectious bronchitis virus infection</article-title><source>Virology</source><volume>306</volume><year>2003</year><fpage>376</fpage><lpage>384</lpage><pub-id pub-id-type="pmid">12642110</pub-id></element-citation></ref><ref id="bib6"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Seo</surname><given-names>S.H.</given-names></name><name><surname>Wang</surname><given-names>L.</given-names></name><name><surname>Smith</surname><given-names>R.</given-names></name><name><surname>Collisson</surname><given-names>E.W.</given-names></name></person-group><article-title>The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection</article-title><source>J. Virol.</source><volume>71</volume><year>1997</year><fpage>7889</fpage><lpage>7894</lpage><pub-id pub-id-type="pmid">9311878</pub-id></element-citation></ref><ref id="bib7"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ressing</surname><given-names>M.E.</given-names></name><name><surname>van Driel</surname><given-names>W.J.</given-names></name><name><surname>Celis</surname><given-names>E.</given-names></name><name><surname>Sette</surname><given-names>A.</given-names></name><name><surname>Brandt</surname><given-names>M.P.</given-names></name><name><surname>Hartman</surname><given-names>M.</given-names></name><name><surname>Anholts</surname><given-names>J.D.</given-names></name><name><surname>Schreuder</surname><given-names>G.M.</given-names></name><name><surname>ter Harmsel</surname><given-names>W.B.</given-names></name><name><surname>Fleuren</surname><given-names>G.J.</given-names></name><name><surname>Trimbos</surname><given-names>B.J.</given-names></name><name><surname>Kast</surname><given-names>W.M.</given-names></name><name><surname>Melief</surname><given-names>C.J.</given-names></name></person-group><article-title>Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type 16-positive cervical lesions against a human leukocyte antigen-A<sup>∗</sup>0201-restricted E7-encoded epitope</article-title><source>Cancer Res.</source><volume>56</volume><year>1996</year><fpage>582</fpage><lpage>588</lpage><pub-id pub-id-type="pmid">8564975</pub-id></element-citation></ref><ref id="bib8"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Passoni</surname><given-names>L.</given-names></name><name><surname>Scardino</surname><given-names>A.</given-names></name><name><surname>Bertazzoli</surname><given-names>C.</given-names></name><name><surname>Gallo</surname><given-names>B.</given-names></name><name><surname>Coluccia</surname><given-names>A.M.</given-names></name><name><surname>Lemonnier</surname><given-names>F.A.</given-names></name><name><surname>Kosmatopoulos</surname><given-names>K.</given-names></name><name><surname>Gambacorti-Passerini</surname><given-names>C.</given-names></name></person-group><article-title>ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8<sup>+</sup> T-cell epitopes</article-title><source>Blood</source><volume>99</volume><year>2002</year><fpage>2100</fpage><lpage>2106</lpage><pub-id pub-id-type="pmid">11877285</pub-id></element-citation></ref><ref id="bib9"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wolfel</surname><given-names>T.</given-names></name><name><surname>Van Pel</surname><given-names>A.</given-names></name><name><surname>Brichard</surname><given-names>V.</given-names></name><name><surname>Schneider</surname><given-names>J.</given-names></name><name><surname>Seliger</surname><given-names>B.</given-names></name><name><surname>Meyer zum Buschenfelde</surname><given-names>K.H.</given-names></name><name><surname>Boon</surname><given-names>T.</given-names></name></person-group><article-title>Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes</article-title><source>Eur. J. Immunol.</source><volume>24</volume><year>1994</year><fpage>759</fpage><lpage>764</lpage><pub-id pub-id-type="pmid">8125142</pub-id></element-citation></ref><ref id="bib10"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>S.J.</given-names></name><name><surname>Leng</surname><given-names>C.H.</given-names></name><name><surname>Lien</surname><given-names>S.P.</given-names></name><name><surname>Chi</surname><given-names>H.Y.</given-names></name><name><surname>Huang</surname><given-names>C.Y.</given-names></name><name><surname>Lin.</surname><given-names>C.L.</given-names></name><name><surname>Lian</surname><given-names>W.C.</given-names></name><name><surname>Chen</surname><given-names>C.J.</given-names></name><name><surname>Hsieh</surname><given-names>S.L.</given-names></name><name><surname>Chong</surname><given-names>P.</given-names></name></person-group><article-title>Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates</article-title><source>Vaccine</source><volume>24</volume><year>2006</year><fpage>3100</fpage><lpage>3108</lpage><pub-id pub-id-type="pmid">16494977</pub-id></element-citation></ref><ref id="bib11"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bullock</surname><given-names>T.N.</given-names></name><name><surname>Colella</surname><given-names>T.A.</given-names></name><name><surname>Engelhard</surname><given-names>V.H.</given-names></name></person-group><article-title>The density of peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice</article-title><source>J. Immunol.</source><volume>164</volume><year>2000</year><fpage>2354</fpage><lpage>2361</lpage><pub-id pub-id-type="pmid">10679070</pub-id></element-citation></ref><ref id="bib12"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>C.W.</given-names></name><name><surname>Lee</surname><given-names>J.Y.</given-names></name><name><surname>Tsao</surname><given-names>Y.P.</given-names></name><name><surname>Shen</surname><given-names>C.P.</given-names></name><name><surname>Lai</surname><given-names>H.C.</given-names></name><name><surname>Chen</surname><given-names>S.L.</given-names></name></person-group><article-title>Oral vaccination with recombinant Listeria monocytogenes expressing human Papillomavirus type 16 E7 can regress tumor growth in mice</article-title><source>Int. J. Cancer</source><volume>298</volume><year>2002</year><fpage>805</fpage><lpage>814</lpage></element-citation></ref><ref id="bib13"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>H.</given-names></name><name><surname>Hou</surname><given-names>J.</given-names></name><name><surname>Jiang</surname><given-names>X.</given-names></name><name><surname>Ma</surname><given-names>S.</given-names></name><name><surname>Meng</surname><given-names>M.</given-names></name><name><surname>Wang</surname><given-names>B.</given-names></name><name><surname>Zhang</surname><given-names>M.</given-names></name><name><surname>Zhang</surname><given-names>M.</given-names></name><name><surname>Tang</surname><given-names>X.</given-names></name><name><surname>Zhang</surname><given-names>F.</given-names></name><name><surname>Wan</surname><given-names>T.</given-names></name><name><surname>Li</surname><given-names>N.</given-names></name><name><surname>Yu</surname><given-names>Y.</given-names></name><name><surname>Hu</surname><given-names>H.</given-names></name><name><surname>Yang</surname><given-names>R.</given-names></name><name><surname>He</surname><given-names>W.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Cao</surname><given-names>X.</given-names></name></person-group><article-title>Response of memory CD8<sup>+</sup> T cells to severe acute respiratory syndrome (SARS) coronavirus in recovered SARS patients and healthy individuals</article-title><source>J. Immunol.</source><volume>175</volume><year>2005</year><fpage>591</fpage><lpage>598</lpage><pub-id pub-id-type="pmid">15972696</pub-id></element-citation></ref><ref id="bib14"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>M.</given-names></name><name><surname>Chu</surname><given-names>C.C.</given-names></name><name><surname>Lee</surname><given-names>H.L.</given-names></name><name><surname>Chang</surname><given-names>S.L.</given-names></name><name><surname>Ohashi</surname><given-names>J.</given-names></name><name><surname>Tokunaga</surname><given-names>K.</given-names></name><name><surname>Akaza</surname><given-names>T.</given-names></name><name><surname>Juji</surname><given-names>T.</given-names></name></person-group><article-title>Heterogeneity of Taiwan’s indigenous population: possible relation to prehistoric Mongoloid dispersals</article-title><source>Tissue Antigens</source><volume>55</volume><year>2000</year><fpage>1</fpage><lpage>9</lpage></element-citation></ref><ref id="bib15"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kaufmann</surname><given-names>A.M.</given-names></name><name><surname>Nieland</surname><given-names>J.</given-names></name><name><surname>Schinz</surname><given-names>M.</given-names></name><name><surname>Nonn</surname><given-names>M.</given-names></name><name><surname>Gabelsberger</surname><given-names>J.</given-names></name><name><surname>Meissner</surname><given-names>H.</given-names></name><name><surname>Muller</surname><given-names>R.T.</given-names></name><name><surname>Jochmus</surname><given-names>I.</given-names></name><name><surname>Gissmann</surname><given-names>L.</given-names></name><name><surname>Schneider</surname><given-names>A.</given-names></name><name><surname>Durst</surname><given-names>M.</given-names></name></person-group><article-title>HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination</article-title><source>Int. J. Cancer</source><volume>92</volume><year>2001</year><fpage>285</fpage><lpage>293</lpage><pub-id pub-id-type="pmid">11291058</pub-id></element-citation></ref><ref id="bib16"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hsueh</surname><given-names>P.R.</given-names></name><name><surname>Yang</surname><given-names>P.C.</given-names></name></person-group><article-title>Severe acute respiratory syndrome epidemic in Taiwan</article-title><source>J. Microbiol. Immunol. Infect.</source><volume>38</volume><year>2005</year><fpage>82</fpage><lpage>88</lpage><pub-id pub-id-type="pmid">15843851</pub-id></element-citation></ref><ref id="bib17"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vigneau</surname><given-names>S.</given-names></name><name><surname>Rohrlich</surname><given-names>P.S.</given-names></name><name><surname>Brahic</surname><given-names>M.</given-names></name><name><surname>Bureau</surname><given-names>J.F.</given-names></name></person-group><article-title>Tmevpg1, a candidate gene for the control of Theiler’s virus persistence, could be implicated in the regulation of gamma interferon</article-title><source>J. Virol.</source><volume>77</volume><year>2003</year><fpage>5632</fpage><lpage>5638</lpage><pub-id pub-id-type="pmid">12719555</pub-id></element-citation></ref><ref id="bib18"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>Y.F.</given-names></name><name><surname>Lin</surname><given-names>C.W.</given-names></name><name><surname>Tsao</surname><given-names>Y.P.</given-names></name><name><surname>Chen</surname><given-names>S.L.</given-names></name></person-group><article-title>Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumor growth in C57BL/6 mice</article-title><source>J. Virol.</source><volume>78</volume><year>2004</year><fpage>1333</fpage><lpage>1343</lpage><pub-id pub-id-type="pmid">14722288</pub-id></element-citation></ref><ref id="bib19"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Evans</surname><given-names>C.</given-names></name><name><surname>Bauer</surname><given-names>S.</given-names></name><name><surname>Grubert</surname><given-names>T.</given-names></name><name><surname>Brucker</surname><given-names>C.</given-names></name><name><surname>Baur</surname><given-names>S.</given-names></name><name><surname>Heeg</surname><given-names>K.</given-names></name><name><surname>Wagner</surname><given-names>H.</given-names></name><name><surname>Lipford</surname><given-names>G.B.</given-names></name></person-group><article-title>HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions</article-title><source>Cancer Immunol. Immunother.</source><volume>42</volume><year>1996</year><fpage>151</fpage><lpage>160</lpage><pub-id pub-id-type="pmid">8640843</pub-id></element-citation></ref><ref id="bib20"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nakagawa</surname><given-names>M.</given-names></name><name><surname>Stites</surname><given-names>D.P.</given-names></name><name><surname>Farhat</surname><given-names>S.</given-names></name><name><surname>Sisler</surname><given-names>J.R.</given-names></name><name><surname>Moss</surname><given-names>B.</given-names></name><name><surname>Kong</surname><given-names>F.</given-names></name><name><surname>Moscicki</surname><given-names>A.B.</given-names></name><name><surname>Palefsky</surname><given-names>J.M.</given-names></name></person-group><article-title>Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia</article-title><source>J. Infect. Dis.</source><volume>175</volume><year>1997</year><fpage>927</fpage><lpage>931</lpage><pub-id pub-id-type="pmid">9086151</pub-id></element-citation></ref><ref id="bib21"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Borysiewicz</surname><given-names>L.K.</given-names></name><name><surname>Fiander</surname><given-names>A.</given-names></name><name><surname>Nimako</surname><given-names>M.</given-names></name><name><surname>Man</surname><given-names>S.</given-names></name><name><surname>Wilkinson</surname><given-names>G.W.</given-names></name><name><surname>Westmoreland</surname><given-names>D.</given-names></name><name><surname>Evans</surname><given-names>A.S.</given-names></name><name><surname>Adams</surname><given-names>M.</given-names></name><name><surname>Stacey</surname><given-names>S.N.</given-names></name><name><surname>Boursnell</surname><given-names>M.E.</given-names></name><name><surname>Rutherford</surname><given-names>E.</given-names></name><name><surname>Hickling</surname><given-names>J.K.</given-names></name><name><surname>Inglis</surname><given-names>S.C.</given-names></name></person-group><article-title>A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer</article-title><source>Lancet</source><volume>347</volume><year>1996</year><fpage>1523</fpage><lpage>1527</lpage><pub-id pub-id-type="pmid">8684105</pub-id></element-citation></ref><ref id="bib22"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schreurs</surname><given-names>M.W.</given-names></name><name><surname>Scholten</surname><given-names>K.B.</given-names></name><name><surname>Kueter</surname><given-names>E.W.</given-names></name><name><surname>Ruizendaal</surname><given-names>J.J.</given-names></name><name><surname>Meijer</surname><given-names>C.J.</given-names></name><name><surname>Hooijberg</surname><given-names>E.</given-names></name></person-group><article-title>In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones</article-title><source>J. Immunol.</source><volume>171</volume><year>2003</year><fpage>2912</fpage><lpage>2921</lpage><pub-id pub-id-type="pmid">12960314</pub-id></element-citation></ref><ref id="bib23"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rivoltini</surname><given-names>L.</given-names></name><name><surname>Loftus</surname><given-names>D.J.</given-names></name><name><surname>Barracchini</surname><given-names>K.</given-names></name><name><surname>Arienti</surname><given-names>F.</given-names></name><name><surname>Mazzocchi</surname><given-names>A.</given-names></name><name><surname>Biddison</surname><given-names>W.E.</given-names></name><name><surname>Salgaller</surname><given-names>M.L.</given-names></name><name><surname>Appella</surname><given-names>E.</given-names></name><name><surname>Parmiani</surname><given-names>G.</given-names></name><name><surname>Marincola</surname><given-names>F.M.</given-names></name></person-group><article-title>Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy</article-title><source>J. Immunol.</source><volume>156</volume><year>1996</year><fpage>3882</fpage><lpage>3891</lpage><pub-id pub-id-type="pmid">8621927</pub-id></element-citation></ref><ref id="bib24"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><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><name><surname>Li</surname><given-names>N.</given-names></name><name><surname>Zhou</surname><given-names>X.</given-names></name><name><surname>Wu</surname><given-names>Y.</given-names></name><name><surname>Yang</surname><given-names>F.</given-names></name><name><surname>Yu</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Yang</surname><given-names>R.</given-names></name><name><surname>Cao</surname><given-names>X.</given-names></name></person-group><article-title>Identification of an HLA-A∗0201-restricted CD8<sup>+</sup> T-cell epitope SSp-1 of SARS-CoV spike protein</article-title><source>Blood</source><volume>104</volume><year>2004</year><fpage>200</fpage><lpage>206</lpage><pub-id pub-id-type="pmid">15016646</pub-id></element-citation></ref><ref id="bib25"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><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><name><surname>Pang</surname><given-names>X.W.</given-names></name><name><surname>He</surname><given-names>X.Y.</given-names></name><name><surname>Zhang</surname><given-names>H.G.</given-names></name><name><surname>Ng</surname><given-names>J.N.</given-names></name><name><surname>Cheng</surname><given-names>C.S.</given-names></name><name><surname>Yu</surname><given-names>J.</given-names></name><name><surname>Meng</surname><given-names>L.</given-names></name><name><surname>Yang</surname><given-names>R.F.</given-names></name><name><surname>Lai</surname><given-names>S.T.</given-names></name><name><surname>Guo</surname><given-names>Z.H.</given-names></name><name><surname>Xie</surname><given-names>Y.</given-names></name><name><surname>Chen</surname><given-names>W.F.</given-names></name></person-group><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><year>2004</year><fpage>5612</fpage><lpage>5618</lpage><pub-id pub-id-type="pmid">15140958</pub-id></element-citation></ref><ref id="bib26"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huang</surname><given-names>L.R.</given-names></name><name><surname>Chiu</surname><given-names>C.M.</given-names></name><name><surname>Yeh</surname><given-names>S.H.</given-names></name><name><surname>Huang</surname><given-names>W.H.</given-names></name><name><surname>Hsue</surname><given-names>P.R.</given-names></name><name><surname>Yang</surname><given-names>W.Z.</given-names></name><name><surname>Yang</surname><given-names>J.Y.</given-names></name><name><surname>Su</surname><given-names>I.J.</given-names></name><name><surname>Chang</surname><given-names>S.C.</given-names></name><name><surname>Chen</surname><given-names>P.J.</given-names></name></person-group><article-title>Evaluation of antibody responses against SARS coronaviral nucleocapsid or spike proteins by immunoblotting or ELISA</article-title><source>J. Med. Virol.</source><volume>73</volume><year>2004</year><fpage>338</fpage><lpage>346</lpage><pub-id pub-id-type="pmid">15170626</pub-id></element-citation></ref><ref id="bib27"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Leung</surname><given-names>D.T.</given-names></name><name><surname>Tam</surname><given-names>F.C.</given-names></name><name><surname>Ma</surname><given-names>C.H.</given-names></name><name><surname>Chan</surname><given-names>P.K.</given-names></name><name><surname>Cheung</surname><given-names>J.L.</given-names></name><name><surname>Niu</surname><given-names>H.</given-names></name><name><surname>Tam</surname><given-names>J.S.</given-names></name><name><surname>Lim</surname><given-names>P.L.</given-names></name></person-group><article-title>Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid</article-title><source>J. Infect. Dis.</source><volume>190</volume><year>2004</year><fpage>379</fpage><lpage>386</lpage><pub-id pub-id-type="pmid">15216476</pub-id></element-citation></ref><ref id="bib28"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Daginakatte</surname><given-names>G.C.</given-names></name><name><surname>Chard-Bergstrom</surname><given-names>C.</given-names></name><name><surname>Andrews</surname><given-names>G.A.</given-names></name><name><surname>Kapil</surname><given-names>S.</given-names></name></person-group><article-title>Production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus</article-title><source>Clin. Diagn. Lab. Immunol.</source><volume>6</volume><year>1999</year><fpage>341</fpage><lpage>344</lpage><pub-id pub-id-type="pmid">10225833</pub-id></element-citation></ref><ref id="bib29"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ndifuna</surname><given-names>A.</given-names></name><name><surname>Waters</surname><given-names>A.K.</given-names></name><name><surname>Zhou</surname><given-names>M.</given-names></name><name><surname>Collisson</surname><given-names>E.W.</given-names></name></person-group><article-title>Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus</article-title><source>J. Virol. Methods</source><volume>70</volume><year>1998</year><fpage>37</fpage><lpage>44</lpage><pub-id pub-id-type="pmid">9506811</pub-id></element-citation></ref><ref id="bib30"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gao</surname><given-names>W.</given-names></name><name><surname>Tamin</surname><given-names>A.</given-names></name><name><surname>Soloff</surname><given-names>A.</given-names></name><name><surname>Aiuto</surname><given-names>L.D.</given-names></name><name><surname>Nwanegbo</surname><given-names>E.</given-names></name><name><surname>Robbins</surname><given-names>P.D.</given-names></name><name><surname>Bellini</surname><given-names>W.J.</given-names></name><name><surname>Barratt-Boyes</surname><given-names>S.</given-names></name><name><surname>Gambotto</surname><given-names>A.</given-names></name></person-group><article-title>Effects of a SARS-associated coronavirus vaccine in monkeys</article-title><source>Lancet</source><year>2003</year><fpage>1895</fpage><lpage>1896</lpage><pub-id pub-id-type="pmid">14667748</pub-id></element-citation></ref><ref id="bib31"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jin</surname><given-names>H.</given-names></name><name><surname>Xiao</surname><given-names>C.</given-names></name><name><surname>Chen</surname><given-names>Z.</given-names></name><name><surname>Kang</surname><given-names>Y.</given-names></name><name><surname>Ma</surname><given-names>Y.</given-names></name><name><surname>Zhu</surname><given-names>K.</given-names></name><name><surname>Xie</surname><given-names>Q.</given-names></name><name><surname>Tu</surname><given-names>Y.</given-names></name><name><surname>Yu</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>B.</given-names></name></person-group><article-title>Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice</article-title><source>Biochem. Biophys. Res. Commun.</source><volume>328</volume><year>2005</year><fpage>979</fpage><lpage>986</lpage><pub-id pub-id-type="pmid">15707974</pub-id></element-citation></ref><ref id="bib32"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><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><name><surname>Icenogle</surname><given-names>J.P.</given-names></name><name><surname>Penaranda</surname><given-names>S.</given-names></name><name><surname>Bankamp</surname><given-names>B.</given-names></name><name><surname>Maher</surname><given-names>K.</given-names></name><name><surname>Chen</surname><given-names>M.H.</given-names></name><name><surname>Tong</surname><given-names>S.</given-names></name><name><surname>Tamin</surname><given-names>A.</given-names></name><name><surname>Lowe</surname><given-names>L.</given-names></name><name><surname>Frace</surname><given-names>M.</given-names></name><name><surname>DeRisi</surname><given-names>J.L.</given-names></name><name><surname>Chen</surname><given-names>Q.</given-names></name><name><surname>Wang</surname><given-names>D.</given-names></name><name><surname>Erdman</surname><given-names>D.D.</given-names></name><name><surname>Peret</surname><given-names>T.C.</given-names></name><name><surname>Burns</surname><given-names>C.</given-names></name><name><surname>Ksiazek</surname><given-names>T.G.</given-names></name><name><surname>Rollin</surname><given-names>P.E.</given-names></name><name><surname>Sanchez</surname><given-names>A.</given-names></name><name><surname>Liffick</surname><given-names>S.</given-names></name><name><surname>Holloway</surname><given-names>B.</given-names></name><name><surname>Limor</surname><given-names>J.</given-names></name><name><surname>McCaustland</surname><given-names>K.</given-names></name><name><surname>Olsen-Rasmussen</surname><given-names>M.</given-names></name><name><surname>Fouchier</surname><given-names>R.</given-names></name><name><surname>Gunther</surname><given-names>S.</given-names></name><name><surname>Osterhaus</surname><given-names>A.D.</given-names></name><name><surname>Drosten</surname><given-names>C.</given-names></name><name><surname>Pallansch</surname><given-names>M.A.</given-names></name><name><surname>Anderson</surname><given-names>L.J.</given-names></name><name><surname>Bellini</surname><given-names>W.J.</given-names></name></person-group><article-title>Characterization of a novel coronavirus associated with severe acute respiratory syndrome</article-title><source>Science</source><volume>300</volume><year>2003</year><fpage>1394</fpage><lpage>1399</lpage><pub-id pub-id-type="pmid">12730500</pub-id></element-citation></ref><ref id="bib33"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Buchholz</surname><given-names>U.J.</given-names></name><name><surname>Bukreyev</surname><given-names>A.</given-names></name><name><surname>Yang</surname><given-names>L.</given-names></name><name><surname>Lamirande</surname><given-names>E.W.</given-names></name><name><surname>Murphy</surname><given-names>B.R.</given-names></name><name><surname>Subbarao</surname><given-names>K.</given-names></name><name><surname>Collins</surname><given-names>P.L.</given-names></name></person-group><article-title>Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity</article-title><source>Proc. Natl. Acad. Sci. USA</source><volume>101</volume><year>2004</year><fpage>9804</fpage><lpage>9809</lpage><pub-id pub-id-type="pmid">15210961</pub-id></element-citation></ref><ref id="bib34"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>He</surname><given-names>Y.</given-names></name><name><surname>Zhou</surname><given-names>Y.</given-names></name><name><surname>Liu</surname><given-names>S.</given-names></name><name><surname>Kou</surname><given-names>Z.</given-names></name><name><surname>Li</surname><given-names>W.</given-names></name><name><surname>Farzan</surname><given-names>M.</given-names></name><name><surname>Jiang</surname><given-names>S.</given-names></name></person-group><article-title>Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine</article-title><source>Biochem. Biophys. Res. Commun.</source><volume>324</volume><year>2004</year><fpage>773</fpage><lpage>781</lpage><pub-id pub-id-type="pmid">15474494</pub-id></element-citation></ref><ref id="bib35"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sui</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>W.</given-names></name><name><surname>Murakami</surname><given-names>A.</given-names></name><name><surname>Tamin</surname><given-names>A.</given-names></name><name><surname>Matthews</surname><given-names>L.J.</given-names></name><name><surname>Wong</surname><given-names>S.K.</given-names></name><name><surname>Moore</surname><given-names>M.J.</given-names></name><name><surname>Tallarico</surname><given-names>A.S.</given-names></name><name><surname>Olurinde</surname><given-names>M.</given-names></name><name><surname>Choe</surname><given-names>H.</given-names></name><name><surname>Anderson</surname><given-names>L.J.</given-names></name><name><surname>Bellini</surname><given-names>W.J.</given-names></name><name><surname>Farzan</surname><given-names>M.</given-names></name><name><surname>Marasco</surname><given-names>W.A.</given-names></name></person-group><article-title>Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association</article-title><source>Proc. Natl. Acad. Sci. USA</source><volume>101</volume><year>2004</year></element-citation></ref><ref id="bib36"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><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></person-group><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><year>2004</year><fpage>3197</fpage><lpage>3201</lpage><pub-id pub-id-type="pmid">14670965</pub-id></element-citation></ref><ref id="bib37"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hua</surname><given-names>R.</given-names></name><name><surname>Zhou</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>Y.</given-names></name><name><surname>Hua</surname><given-names>Y.</given-names></name><name><surname>Tong</surname><given-names>G.</given-names></name></person-group><article-title>Identification of two antigenic epitopes on SARS-CoV spike protein</article-title><source>Biochem. Biophys. Res. Commun.</source><volume>319</volume><year>2004</year><fpage>929</fpage><lpage>935</lpage><pub-id pub-id-type="pmid">15184071</pub-id></element-citation></ref><ref id="bib38"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Krokhin</surname><given-names>O.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Andonov</surname><given-names>A.</given-names></name><name><surname>Feldmann</surname><given-names>H.</given-names></name><name><surname>Flick</surname><given-names>R.</given-names></name><name><surname>Jones</surname><given-names>S.</given-names></name><name><surname>Stroeher</surname><given-names>U.</given-names></name><name><surname>Bastien</surname><given-names>N.</given-names></name><name><surname>Dasuri</surname><given-names>K.V.</given-names></name><name><surname>Cheng</surname><given-names>K.</given-names></name><name><surname>Simonsen</surname><given-names>J.N.</given-names></name><name><surname>Perreault</surname><given-names>H.</given-names></name><name><surname>Wilkins</surname><given-names>J.</given-names></name><name><surname>Ens</surname><given-names>W.</given-names></name><name><surname>Plummer</surname><given-names>F.</given-names></name><name><surname>Standing</surname><given-names>K.G.</given-names></name></person-group><article-title>Mass spectrometric characterization of proteins from the SARS virus: a preliminary report</article-title><source>Mol. Cell Proteomics</source><volume>2</volume><year>2003</year><fpage>346</fpage><lpage>356</lpage><pub-id pub-id-type="pmid">12775768</pub-id></element-citation></ref><ref id="bib39"><label>39</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhong</surname><given-names>X.</given-names></name><name><surname>Yang</surname><given-names>H.</given-names></name><name><surname>Guo</surname><given-names>Z.F.</given-names></name><name><surname>Sin</surname><given-names>W.Y.</given-names></name><name><surname>Chen</surname><given-names>W.</given-names></name><name><surname>Xu</surname><given-names>J.</given-names></name><name><surname>Fu</surname><given-names>L.</given-names></name><name><surname>Wu</surname><given-names>J.</given-names></name><name><surname>Mak</surname><given-names>C.K.</given-names></name><name><surname>Cheng</surname><given-names>C.S.</given-names></name><name><surname>Yang</surname><given-names>Y.</given-names></name><name><surname>Cao</surname><given-names>S.</given-names></name><name><surname>Wong</surname><given-names>T.Y.</given-names></name><name><surname>Lai</surname><given-names>S.T.</given-names></name><name><surname>Xie</surname><given-names>Y.</given-names></name><name><surname>Guo</surname><given-names>Z.</given-names></name></person-group><article-title>B-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein</article-title><source>J. Virol.</source><volume>79</volume><year>2005</year><fpage>3401</fpage><lpage>3408</lpage><pub-id pub-id-type="pmid">15731234</pub-id></element-citation></ref><ref id="bib40"><label>40</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>Z.</given-names></name><name><surname>Zhang</surname><given-names>L.</given-names></name><name><surname>Qin</surname><given-names>C.</given-names></name><name><surname>Ba</surname><given-names>L.</given-names></name><name><surname>Yi</surname><given-names>C.E.</given-names></name><name><surname>Zhang</surname><given-names>F.</given-names></name><name><surname>Wei</surname><given-names>Q.</given-names></name><name><surname>He</surname><given-names>T.</given-names></name><name><surname>Yu</surname><given-names>W.</given-names></name><name><surname>Yu</surname><given-names>J.</given-names></name><name><surname>Gao</surname><given-names>H.</given-names></name><name><surname>Tu</surname><given-names>X.</given-names></name><name><surname>Gettie</surname><given-names>A.</given-names></name><name><surname>Farzan</surname><given-names>M.</given-names></name><name><surname>Yuen</surname><given-names>K.Y.</given-names></name><name><surname>Ho</surname><given-names>D.D.</given-names></name></person-group><article-title>Recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region</article-title><source>J. Virol.</source><volume>79</volume><year>2005</year><fpage>2678</fpage><lpage>2688</lpage><pub-id pub-id-type="pmid">15708987</pub-id></element-citation></ref></ref-list><sec id="app1" sec-type="supplementary-material"><label>Appendix A</label><title>Supplementary data</title><p id="para.0110"><supplementary-material content-type="local-data" id="e-component.0010"><caption><title>Supplementary data</title></caption><media xlink:href="mmc1.doc"></media></supplementary-material></p></sec><ack><title>Acknowledgments</title><p>We are grateful to Dr. Kernick James Deen for editing English and to Ching-Jen Yang for his technical assistance. This work was supported by National Science Council Grant NSC 92-2751-B-016-006-Y, NSC 91-2320-B-016-019, NSC 91-3112-B-016-003-M51, and National Health Research Institute NHRI-EX91-9013BL.</p></ack><fn-group><fn id="section.0045" fn-type="supplementary-material"><label>Appendix A</label><p id="para.0105">Supplementary data associated with this article can be found, in the online version, at <ext-link ext-link-type="doi" xlink:href="10.1016/j.bbrc.2006.03.152">doi:10.1016/j.bbrc.2006.03.152</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000E28 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000E28 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= SrasV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.33. |  |