Amino acids 1055 to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents.
Identifieur interne : 000D96 ( Ncbi/Checkpoint ); précédent : 000D95; suivant : 000D97Amino acids 1055 to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents.
Auteurs : Choong-Tat Keng ; Aihua Zhang ; Shuo Shen ; Kuo-Ming Lip ; Burtram C. Fielding ; Timothy H P. Tan ; Chih-Fong Chou ; Chay Boon Loh ; Sifang Wang ; Jianlin Fu ; Xiaoming Yang ; Seng Gee Lim ; Wanjin Hong ; Yee-Joo TanSource :
- Journal of virology [ 0022-538X ] ; 2005.
Descripteurs français
- KwdFr :
- Animaux, Anticorps antiviraux (immunologie), Cartographie épitopique, Cellules cultivées, Clonage moléculaire, Escherichia coli (génétique), Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (génétique), Glycoprotéines membranaires (immunologie), Glycoprotéines membranaires (physiologie), Immunoprécipitation, Lapins, Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (immunologie), Protéines de fusion recombinantes (isolement et purification), Protéines de l'enveloppe virale (génétique), Protéines de l'enveloppe virale (immunologie), Protéines de l'enveloppe virale (physiologie), Technique d'immunofluorescence, Technique de Western, Tests de neutralisation, Virus du SRAS (immunologie), Épitopes (génétique), Épitopes (immunologie).
- MESH :
- génétique : Escherichia coli, Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Épitopes.
- immunologie : Anticorps antiviraux, Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Virus du SRAS, Épitopes.
- isolement et purification : Protéines de fusion recombinantes.
- physiologie : Glycoprotéines membranaires, Protéines de l'enveloppe virale.
- Animaux, Cartographie épitopique, Cellules cultivées, Clonage moléculaire, Glycoprotéine de spicule des coronavirus, Immunoprécipitation, Lapins, Technique d'immunofluorescence, Technique de Western, Tests de neutralisation.
English descriptors
- KwdEn :
- Animals, Antibodies, Viral (immunology), Blotting, Western, Cells, Cultured, Chlorocebus aethiops, Cloning, Molecular, Epitope Mapping, Epitopes (genetics), Epitopes (immunology), Escherichia coli (genetics), Fluorescent Antibody Technique, Immunoprecipitation, Membrane Glycoproteins (genetics), Membrane Glycoproteins (immunology), Membrane Glycoproteins (physiology), Neutralization Tests, Rabbits, Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (immunology), Recombinant Fusion Proteins (isolation & purification), SARS Virus (immunology), Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (genetics), Viral Envelope Proteins (immunology), Viral Envelope Proteins (physiology).
- MESH :
- chemical , genetics : Epitopes, Membrane Glycoproteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- chemical , immunology : Antibodies, Viral, Epitopes, Membrane Glycoproteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- genetics : Escherichia coli.
- immunology : SARS Virus.
- chemical , isolation & purification : Recombinant Fusion Proteins.
- chemical , physiology : Membrane Glycoproteins, Viral Envelope Proteins.
- Animals, Blotting, Western, Cells, Cultured, Chlorocebus aethiops, Cloning, Molecular, Epitope Mapping, Fluorescent Antibody Technique, Immunoprecipitation, Neutralization Tests, Rabbits, Spike Glycoprotein, Coronavirus.
Abstract
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
DOI: 10.1128/JVI.79.6.3289-3296.2005
PubMed: 15731223
Affiliations:
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Fielding</name></author><author><name sortKey="Tan, Timothy H P" sort="Tan, Timothy H P" uniqKey="Tan T" first="Timothy H P" last="Tan">Timothy H P. Tan</name></author><author><name sortKey="Chou, Chih Fong" sort="Chou, Chih Fong" uniqKey="Chou C" first="Chih-Fong" last="Chou">Chih-Fong Chou</name></author><author><name sortKey="Loh, Chay Boon" sort="Loh, Chay Boon" uniqKey="Loh C" first="Chay Boon" last="Loh">Chay Boon Loh</name></author><author><name sortKey="Wang, Sifang" sort="Wang, Sifang" uniqKey="Wang S" first="Sifang" last="Wang">Sifang Wang</name></author><author><name sortKey="Fu, Jianlin" sort="Fu, Jianlin" uniqKey="Fu J" first="Jianlin" last="Fu">Jianlin Fu</name></author><author><name sortKey="Yang, Xiaoming" sort="Yang, Xiaoming" uniqKey="Yang X" first="Xiaoming" last="Yang">Xiaoming Yang</name></author><author><name sortKey="Lim, Seng Gee" sort="Lim, Seng Gee" uniqKey="Lim S" first="Seng Gee" last="Lim">Seng Gee Lim</name></author><author><name sortKey="Hong, Wanjin" sort="Hong, Wanjin" uniqKey="Hong W" first="Wanjin" last="Hong">Wanjin Hong</name></author><author><name sortKey="Tan, Yee Joo" sort="Tan, Yee Joo" uniqKey="Tan Y" first="Yee-Joo" last="Tan">Yee-Joo Tan</name></author></analytic><series><title level="j">Journal of virology</title><idno type="ISSN">0022-538X</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antibodies, Viral (immunology)</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Chlorocebus aethiops</term><term>Cloning, Molecular</term><term>Epitope Mapping</term><term>Epitopes (genetics)</term><term>Epitopes (immunology)</term><term>Escherichia coli (genetics)</term><term>Fluorescent Antibody Technique</term><term>Immunoprecipitation</term><term>Membrane Glycoproteins (genetics)</term><term>Membrane Glycoproteins (immunology)</term><term>Membrane Glycoproteins (physiology)</term><term>Neutralization Tests</term><term>Rabbits</term><term>Recombinant Fusion Proteins (genetics)</term><term>Recombinant Fusion Proteins (immunology)</term><term>Recombinant Fusion Proteins (isolation & purification)</term><term>SARS Virus (immunology)</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (genetics)</term><term>Viral Envelope Proteins (immunology)</term><term>Viral Envelope Proteins (physiology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Anticorps antiviraux (immunologie)</term><term>Cartographie épitopique</term><term>Cellules cultivées</term><term>Clonage moléculaire</term><term>Escherichia coli (génétique)</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires (génétique)</term><term>Glycoprotéines membranaires (immunologie)</term><term>Glycoprotéines membranaires (physiologie)</term><term>Immunoprécipitation</term><term>Lapins</term><term>Protéines de fusion recombinantes (génétique)</term><term>Protéines de fusion recombinantes (immunologie)</term><term>Protéines de fusion recombinantes (isolement et purification)</term><term>Protéines de l'enveloppe virale (génétique)</term><term>Protéines de l'enveloppe virale (immunologie)</term><term>Protéines de l'enveloppe virale (physiologie)</term><term>Technique d'immunofluorescence</term><term>Technique de Western</term><term>Tests de neutralisation</term><term>Virus du SRAS (immunologie)</term><term>Épitopes (génétique)</term><term>Épitopes (immunologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Epitopes</term><term>Membrane Glycoproteins</term><term>Recombinant Fusion Proteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antibodies, Viral</term><term>Epitopes</term><term>Membrane Glycoproteins</term><term>Recombinant Fusion Proteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Escherichia coli</term><term>Glycoprotéines membranaires</term><term>Protéines de fusion recombinantes</term><term>Protéines de l'enveloppe virale</term><term>Épitopes</term></keywords><keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Anticorps antiviraux</term><term>Glycoprotéines membranaires</term><term>Protéines de fusion recombinantes</term><term>Protéines de l'enveloppe virale</term><term>Virus du SRAS</term><term>Épitopes</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines de fusion recombinantes</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Glycoprotéines membranaires</term><term>Protéines de l'enveloppe virale</term></keywords><keywords scheme="MESH" type="chemical" qualifier="physiology" xml:lang="en"><term>Membrane Glycoproteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Chlorocebus aethiops</term><term>Cloning, Molecular</term><term>Epitope Mapping</term><term>Fluorescent Antibody Technique</term><term>Immunoprecipitation</term><term>Neutralization Tests</term><term>Rabbits</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cartographie épitopique</term><term>Cellules cultivées</term><term>Clonage moléculaire</term><term>Glycoprotéine de spicule des coronavirus</term><term>Immunoprécipitation</term><term>Lapins</term><term>Technique d'immunofluorescence</term><term>Technique de Western</term><term>Tests de neutralisation</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.</div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Chou, Chih Fong" sort="Chou, Chih Fong" uniqKey="Chou C" first="Chih-Fong" last="Chou">Chih-Fong Chou</name><name sortKey="Fielding, Burtram C" sort="Fielding, Burtram C" uniqKey="Fielding B" first="Burtram C" last="Fielding">Burtram C. Fielding</name><name sortKey="Fu, Jianlin" sort="Fu, Jianlin" uniqKey="Fu J" first="Jianlin" last="Fu">Jianlin Fu</name><name sortKey="Hong, Wanjin" sort="Hong, Wanjin" uniqKey="Hong W" first="Wanjin" last="Hong">Wanjin Hong</name><name sortKey="Keng, Choong Tat" sort="Keng, Choong Tat" uniqKey="Keng C" first="Choong-Tat" last="Keng">Choong-Tat Keng</name><name sortKey="Lim, Seng Gee" sort="Lim, Seng Gee" uniqKey="Lim S" first="Seng Gee" last="Lim">Seng Gee Lim</name><name sortKey="Lip, Kuo Ming" sort="Lip, Kuo Ming" uniqKey="Lip K" first="Kuo-Ming" last="Lip">Kuo-Ming Lip</name><name sortKey="Loh, Chay Boon" sort="Loh, Chay Boon" uniqKey="Loh C" first="Chay Boon" last="Loh">Chay Boon Loh</name><name sortKey="Shen, Shuo" sort="Shen, Shuo" uniqKey="Shen S" first="Shuo" last="Shen">Shuo Shen</name><name sortKey="Tan, Timothy H P" sort="Tan, Timothy H P" uniqKey="Tan T" first="Timothy H P" last="Tan">Timothy H P. Tan</name><name sortKey="Tan, Yee Joo" sort="Tan, Yee Joo" uniqKey="Tan Y" first="Yee-Joo" last="Tan">Yee-Joo Tan</name><name sortKey="Wang, Sifang" sort="Wang, Sifang" uniqKey="Wang S" first="Sifang" last="Wang">Sifang Wang</name><name sortKey="Yang, Xiaoming" sort="Yang, Xiaoming" uniqKey="Yang X" first="Xiaoming" last="Yang">Xiaoming Yang</name><name sortKey="Zhang, Aihua" sort="Zhang, Aihua" uniqKey="Zhang A" first="Aihua" last="Zhang">Aihua Zhang</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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