A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.
Identifieur interne : 000853 ( PubMed/Corpus ); précédent : 000852; suivant : 000854A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.
Auteurs : Guangyu Zhao ; Lei He ; Shihui Sun ; Hongjie Qiu ; Wanbo Tai ; Jiawei Chen ; Jiangfan Li ; Yuehong Chen ; Yan Guo ; Yufei Wang ; Jian Shang ; Kaiyuan Ji ; Ruiwen Fan ; Enqi Du ; Shibo Jiang ; Fang Li ; Lanying Du ; Yusen ZhouSource :
- Journal of virology [ 1098-5514 ] ; 2018.
English descriptors
- KwdEn :
- Animals, Antibodies, Neutralizing (chemistry), Antibodies, Neutralizing (immunology), Antibodies, Viral (immunology), Binding Sites (immunology), Coronavirus Infections (immunology), Coronavirus Infections (prevention & control), Coronavirus Infections (therapy), Epitopes (metabolism), Humans, Mice, Mice, Inbred BALB C, Middle East Respiratory Syndrome Coronavirus (immunology), Neutralization Tests, Protein Binding, Single-Domain Antibodies (economics), Single-Domain Antibodies (immunology), Single-Domain Antibodies (isolation & purification), Single-Domain Antibodies (metabolism), Spike Glycoprotein, Coronavirus (genetics), Spike Glycoprotein, Coronavirus (immunology).
- MESH :
- chemical , chemistry : Antibodies, Neutralizing.
- chemical , economics : Single-Domain Antibodies.
- chemical , genetics : Spike Glycoprotein, Coronavirus.
- chemical , immunology : Antibodies, Neutralizing, Antibodies, Viral, Single-Domain Antibodies, Spike Glycoprotein, Coronavirus.
- immunology : Binding Sites, Coronavirus Infections, Middle East Respiratory Syndrome Coronavirus.
- chemical , isolation & purification : Single-Domain Antibodies.
- chemical , metabolism : Epitopes, Single-Domain Antibodies.
- prevention & control : Coronavirus Infections.
- therapy : Coronavirus Infections.
- Animals, Humans, Mice, Mice, Inbred BALB C, Neutralization Tests, Protein Binding.
Abstract
The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.
DOI: 10.1128/JVI.00837-18
PubMed: 29950421
Links to Exploration step
pubmed:29950421Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.</title><author><name sortKey="Zhao, Guangyu" sort="Zhao, Guangyu" uniqKey="Zhao G" first="Guangyu" last="Zhao">Guangyu Zhao</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="He, Lei" sort="He, Lei" uniqKey="He L" first="Lei" last="He">Lei He</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Sun, Shihui" sort="Sun, Shihui" uniqKey="Sun S" first="Shihui" last="Sun">Shihui Sun</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Qiu, Hongjie" sort="Qiu, Hongjie" uniqKey="Qiu H" first="Hongjie" last="Qiu">Hongjie Qiu</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Tai, Wanbo" sort="Tai, Wanbo" uniqKey="Tai W" first="Wanbo" last="Tai">Wanbo Tai</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Chen, Jiawei" sort="Chen, Jiawei" uniqKey="Chen J" first="Jiawei" last="Chen">Jiawei Chen</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Li, Jiangfan" sort="Li, Jiangfan" uniqKey="Li J" first="Jiangfan" last="Li">Jiangfan Li</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Chen, Yuehong" sort="Chen, Yuehong" uniqKey="Chen Y" first="Yuehong" last="Chen">Yuehong Chen</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Guo, Yan" sort="Guo, Yan" uniqKey="Guo Y" first="Yan" last="Guo">Yan Guo</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Wang, Yufei" sort="Wang, Yufei" uniqKey="Wang Y" first="Yufei" last="Wang">Yufei Wang</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Shang, Jian" sort="Shang, Jian" uniqKey="Shang J" first="Jian" last="Shang">Jian Shang</name><affiliation><nlm:affiliation>Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Ji, Kaiyuan" sort="Ji, Kaiyuan" uniqKey="Ji K" first="Kaiyuan" last="Ji">Kaiyuan Ji</name><affiliation><nlm:affiliation>ShanXi Agricultural University, Shanxi, China.</nlm:affiliation></affiliation></author><author><name sortKey="Fan, Ruiwen" sort="Fan, Ruiwen" uniqKey="Fan R" first="Ruiwen" last="Fan">Ruiwen Fan</name><affiliation><nlm:affiliation>ShanXi Agricultural University, Shanxi, China.</nlm:affiliation></affiliation></author><author><name sortKey="Du, Enqi" sort="Du, Enqi" uniqKey="Du E" first="Enqi" last="Du">Enqi Du</name><affiliation><nlm:affiliation>Northwest A&F University, Shaanxi, China.</nlm:affiliation></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name><affiliation><nlm:affiliation>Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA ldu@nybc.org yszhou@bmi.ac.cn.</nlm:affiliation></affiliation></author><author><name sortKey="Zhou, Yusen" sort="Zhou, Yusen" uniqKey="Zhou Y" first="Yusen" last="Zhou">Yusen Zhou</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China ldu@nybc.org yszhou@bmi.ac.cn.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2018">2018</date><idno type="RBID">pubmed:29950421</idno><idno type="pmid">29950421</idno><idno type="doi">10.1128/JVI.00837-18</idno><idno type="wicri:Area/PubMed/Corpus">000853</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000853</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.</title><author><name sortKey="Zhao, Guangyu" sort="Zhao, Guangyu" uniqKey="Zhao G" first="Guangyu" last="Zhao">Guangyu Zhao</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="He, Lei" sort="He, Lei" uniqKey="He L" first="Lei" last="He">Lei He</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Sun, Shihui" sort="Sun, Shihui" uniqKey="Sun S" first="Shihui" last="Sun">Shihui Sun</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Qiu, Hongjie" sort="Qiu, Hongjie" uniqKey="Qiu H" first="Hongjie" last="Qiu">Hongjie Qiu</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Tai, Wanbo" sort="Tai, Wanbo" uniqKey="Tai W" first="Wanbo" last="Tai">Wanbo Tai</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Chen, Jiawei" sort="Chen, Jiawei" uniqKey="Chen J" first="Jiawei" last="Chen">Jiawei Chen</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Li, Jiangfan" sort="Li, Jiangfan" uniqKey="Li J" first="Jiangfan" last="Li">Jiangfan Li</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Chen, Yuehong" sort="Chen, Yuehong" uniqKey="Chen Y" first="Yuehong" last="Chen">Yuehong Chen</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Guo, Yan" sort="Guo, Yan" uniqKey="Guo Y" first="Yan" last="Guo">Yan Guo</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</nlm:affiliation></affiliation></author><author><name sortKey="Wang, Yufei" sort="Wang, Yufei" uniqKey="Wang Y" first="Yufei" last="Wang">Yufei Wang</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Shang, Jian" sort="Shang, Jian" uniqKey="Shang J" first="Jian" last="Shang">Jian Shang</name><affiliation><nlm:affiliation>Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Ji, Kaiyuan" sort="Ji, Kaiyuan" uniqKey="Ji K" first="Kaiyuan" last="Ji">Kaiyuan Ji</name><affiliation><nlm:affiliation>ShanXi Agricultural University, Shanxi, China.</nlm:affiliation></affiliation></author><author><name sortKey="Fan, Ruiwen" sort="Fan, Ruiwen" uniqKey="Fan R" first="Ruiwen" last="Fan">Ruiwen Fan</name><affiliation><nlm:affiliation>ShanXi Agricultural University, Shanxi, China.</nlm:affiliation></affiliation></author><author><name sortKey="Du, Enqi" sort="Du, Enqi" uniqKey="Du E" first="Enqi" last="Du">Enqi Du</name><affiliation><nlm:affiliation>Northwest A&F University, Shaanxi, China.</nlm:affiliation></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name><affiliation><nlm:affiliation>Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name><affiliation><nlm:affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA ldu@nybc.org yszhou@bmi.ac.cn.</nlm:affiliation></affiliation></author><author><name sortKey="Zhou, Yusen" sort="Zhou, Yusen" uniqKey="Zhou Y" first="Yusen" last="Zhou">Yusen Zhou</name><affiliation><nlm:affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China ldu@nybc.org yszhou@bmi.ac.cn.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Journal of virology</title><idno type="eISSN">1098-5514</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antibodies, Neutralizing (chemistry)</term><term>Antibodies, Neutralizing (immunology)</term><term>Antibodies, Viral (immunology)</term><term>Binding Sites (immunology)</term><term>Coronavirus Infections (immunology)</term><term>Coronavirus Infections (prevention & control)</term><term>Coronavirus Infections (therapy)</term><term>Epitopes (metabolism)</term><term>Humans</term><term>Mice</term><term>Mice, Inbred BALB C</term><term>Middle East Respiratory Syndrome Coronavirus (immunology)</term><term>Neutralization Tests</term><term>Protein Binding</term><term>Single-Domain Antibodies (economics)</term><term>Single-Domain Antibodies (immunology)</term><term>Single-Domain Antibodies (isolation & purification)</term><term>Single-Domain Antibodies (metabolism)</term><term>Spike Glycoprotein, Coronavirus (genetics)</term><term>Spike Glycoprotein, Coronavirus (immunology)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Antibodies, Neutralizing</term></keywords><keywords scheme="MESH" type="chemical" qualifier="economics" xml:lang="en"><term>Single-Domain Antibodies</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antibodies, Neutralizing</term><term>Antibodies, Viral</term><term>Single-Domain Antibodies</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>Binding Sites</term><term>Coronavirus Infections</term><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Single-Domain Antibodies</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Epitopes</term><term>Single-Domain Antibodies</term></keywords><keywords scheme="MESH" qualifier="prevention & control" xml:lang="en"><term>Coronavirus Infections</term></keywords><keywords scheme="MESH" qualifier="therapy" xml:lang="en"><term>Coronavirus Infections</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Humans</term><term>Mice</term><term>Mice, Inbred BALB C</term><term>Neutralization Tests</term><term>Protein Binding</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life <i>in vivo</i>; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.<b>IMPORTANCE</b> Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the <i>in vivo</i> half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">29950421</PMID><DateCompleted><Year>2018</Year><Month>09</Month><Day>10</Day></DateCompleted><DateRevised><Year>2018</Year><Month>12</Month><Day>25</Day></DateRevised><Article PubModel="Electronic-Print"><Journal><ISSN IssnType="Electronic">1098-5514</ISSN><JournalIssue CitedMedium="Internet"><Volume>92</Volume><Issue>18</Issue><PubDate><Year>2018</Year><Month>09</Month><Day>15</Day></PubDate></JournalIssue><Title>Journal of virology</Title><ISOAbbreviation>J. Virol.</ISOAbbreviation></Journal><ArticleTitle>A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.</ArticleTitle><ELocationID EIdType="pii" ValidYN="Y">e00837-18</ELocationID><ELocationID EIdType="doi" ValidYN="Y">10.1128/JVI.00837-18</ELocationID><Abstract><AbstractText>The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life <i>in vivo</i>; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.<b>IMPORTANCE</b> Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the <i>in vivo</i> half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.</AbstractText><CopyrightInformation>Copyright © 2018 Zhao et al.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y" EqualContrib="Y"><LastName>Zhao</LastName><ForeName>Guangyu</ForeName><Initials>G</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y" EqualContrib="Y"><LastName>He</LastName><ForeName>Lei</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y" EqualContrib="Y"><LastName>Sun</LastName><ForeName>Shihui</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Qiu</LastName><ForeName>Hongjie</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tai</LastName><ForeName>Wanbo</ForeName><Initials>W</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Jiawei</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Li</LastName><ForeName>Jiangfan</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chen</LastName><ForeName>Yuehong</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Guo</LastName><ForeName>Yan</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wang</LastName><ForeName>Yufei</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Shang</LastName><ForeName>Jian</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ji</LastName><ForeName>Kaiyuan</ForeName><Initials>K</Initials><AffiliationInfo><Affiliation>ShanXi Agricultural University, Shanxi, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Fan</LastName><ForeName>Ruiwen</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>ShanXi Agricultural University, Shanxi, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Du</LastName><ForeName>Enqi</ForeName><Initials>E</Initials><AffiliationInfo><Affiliation>Northwest A&F University, Shaanxi, China.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Jiang</LastName><ForeName>Shibo</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y" EqualContrib="Y"><LastName>Li</LastName><ForeName>Fang</ForeName><Initials>F</Initials><AffiliationInfo><Affiliation>Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y" EqualContrib="Y"><LastName>Du</LastName><ForeName>Lanying</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA ldu@nybc.org yszhou@bmi.ac.cn.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y" EqualContrib="Y"><LastName>Zhou</LastName><ForeName>Yusen</ForeName><Initials>Y</Initials><AffiliationInfo><Affiliation>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China ldu@nybc.org yszhou@bmi.ac.cn.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>R01 AI089728</GrantID><Acronym>AI</Acronym><Agency>NIAID NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>R01 AI110700</GrantID><Acronym>AI</Acronym><Agency>NIAID NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>R01 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