Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
Identifieur interne : 000990 ( Main/Exploration ); précédent : 000989; suivant : 000991Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
Auteurs : Mun Peak Nyon [Malaisie, États-Unis] ; Lanying Du [États-Unis] ; Chien-Te Kent Tseng [États-Unis] ; Christopher A. Seid [États-Unis] ; Jeroen Pollet [États-Unis] ; Kevin S. Naceanceno [États-Unis] ; Anurodh Agrawal [États-Unis] ; Abdullah Algaissi [États-Unis] ; Bi-Hung Peng [États-Unis] ; Wanbo Tai [États-Unis] ; Shibo Jiang [États-Unis, République populaire de Chine] ; Maria Elena Bottazzi [États-Unis] ; Ulrich Strych [États-Unis] ; Peter J. Hotez [États-Unis]Source :
- Vaccine [ 0264-410X ] ; 2018.
Descripteurs français
- KwdFr :
- Animaux, Antigènes viraux (génétique), Antigènes viraux (immunologie), Cellules CHO, Coronavirus du syndrome respiratoire du Moyen-Orient (immunologie), Cricetulus, Expression des gènes, Fragments Fc des immunoglobulines (génétique), Fragments Fc des immunoglobulines (immunologie), Génie génétique, Immunogénicité des vaccins, Infections à coronavirus (), Infections à coronavirus (immunologie), Maturation post-traductionnelle des protéines, Protéines recombinantes (génétique), Protéines recombinantes (immunologie), Souris, Vaccins antiviraux (immunologie), Vecteurs génétiques (génétique), Épitopes (), Épitopes (immunologie).
- MESH :
- génétique : Antigènes viraux, Fragments Fc des immunoglobulines, Protéines recombinantes, Vecteurs génétiques.
- immunologie : Antigènes viraux, Coronavirus du syndrome respiratoire du Moyen-Orient, Fragments Fc des immunoglobulines, Infections à coronavirus, Protéines recombinantes, Vaccins antiviraux, Épitopes.
- Animaux, Cellules CHO, Cricetulus, Expression des gènes, Génie génétique, Immunogénicité des vaccins, Infections à coronavirus, Maturation post-traductionnelle des protéines, Souris, Épitopes.
English descriptors
- KwdEn :
- Animals, Antigens, Viral (genetics), Antigens, Viral (immunology), CHO Cells, Coronavirus Infections (immunology), Coronavirus Infections (prevention & control), Cricetulus, Epitopes (chemistry), Epitopes (immunology), Gene Expression, Genetic Engineering, Genetic Vectors (genetics), Immunogenicity, Vaccine, Immunoglobulin Fc Fragments (genetics), Immunoglobulin Fc Fragments (immunology), Mice, Middle East Respiratory Syndrome Coronavirus (immunology), Protein Processing, Post-Translational, Recombinant Proteins (genetics), Recombinant Proteins (immunology), Viral Vaccines (immunology).
- MESH :
- chemical , chemistry : Epitopes.
- chemical , genetics : Antigens, Viral, Immunoglobulin Fc Fragments, Recombinant Proteins.
- chemical , immunology : Antigens, Viral, Epitopes, Immunoglobulin Fc Fragments, Recombinant Proteins, Viral Vaccines.
- genetics : Genetic Vectors.
- immunology : Coronavirus Infections, Middle East Respiratory Syndrome Coronavirus.
- prevention & control : Coronavirus Infections.
- Animals, CHO Cells, Cricetulus, Gene Expression, Genetic Engineering, Immunogenicity, Vaccine, Mice, Protein Processing, Post-Translational.
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.
Url:
DOI: 10.1016/j.vaccine.2018.02.065
PubMed: 29496347
PubMed Central: 5860679
Affiliations:
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Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation wicri:level="2"><nlm:aff id="af020">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Lindsley F. 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Hotez</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29496347</idno><idno type="pmc">5860679</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860679</idno><idno type="RBID">PMC:5860679</idno><idno type="doi">10.1016/j.vaccine.2018.02.065</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">001170</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001170</idno><idno type="wicri:Area/Pmc/Curation">001170</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">001170</idno><idno type="wicri:Area/Pmc/Checkpoint">000591</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000591</idno><idno type="wicri:source">PubMed</idno><idno type="RBID">pubmed:29496347</idno><idno type="wicri:Area/PubMed/Corpus">000987</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000987</idno><idno type="wicri:Area/PubMed/Curation">000987</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">000987</idno><idno type="wicri:Area/PubMed/Checkpoint">000935</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">000935</idno><idno type="wicri:Area/Ncbi/Merge">001D45</idno><idno type="wicri:Area/Ncbi/Curation">001D45</idno><idno type="wicri:Area/Ncbi/Checkpoint">001D45</idno><idno type="wicri:doubleKey">0264-410X:2018:Nyon M:engineering:a:stable</idno><idno type="wicri:Area/Main/Merge">000993</idno><idno type="wicri:Area/Main/Curation">000990</idno><idno type="wicri:Area/Main/Exploration">000990</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen</title><author><name sortKey="Nyon, Mun Peak" sort="Nyon, Mun Peak" uniqKey="Nyon M" first="Mun Peak" last="Nyon">Mun Peak Nyon</name><affiliation wicri:level="1"><nlm:aff id="af005">Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur, Malaysia</nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea>Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur</wicri:regionArea><wicri:noRegion>Kuala Lumpur</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name><affiliation wicri:level="2"><nlm:aff id="af020">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Tseng, Chien Te Kent" sort="Tseng, Chien Te Kent" uniqKey="Tseng C" first="Chien-Te Kent" last="Tseng">Chien-Te Kent Tseng</name><affiliation wicri:level="2"><nlm:aff id="af025">Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Seid, Christopher A" sort="Seid, Christopher A" uniqKey="Seid C" first="Christopher A." last="Seid">Christopher A. Seid</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Pollet, Jeroen" sort="Pollet, Jeroen" uniqKey="Pollet J" first="Jeroen" last="Pollet">Jeroen Pollet</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Naceanceno, Kevin S" sort="Naceanceno, Kevin S" uniqKey="Naceanceno K" first="Kevin S." last="Naceanceno">Kevin S. Naceanceno</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Agrawal, Anurodh" sort="Agrawal, Anurodh" uniqKey="Agrawal A" first="Anurodh" last="Agrawal">Anurodh Agrawal</name><affiliation wicri:level="2"><nlm:aff id="af025">Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Algaissi, Abdullah" sort="Algaissi, Abdullah" uniqKey="Algaissi A" first="Abdullah" last="Algaissi">Abdullah Algaissi</name><affiliation wicri:level="2"><nlm:aff id="af025">Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Peng, Bi Hung" sort="Peng, Bi Hung" uniqKey="Peng B" first="Bi-Hung" last="Peng">Bi-Hung Peng</name><affiliation wicri:level="2"><nlm:aff id="af025">Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Tai, Wanbo" sort="Tai, Wanbo" uniqKey="Tai W" first="Wanbo" last="Tai">Wanbo Tai</name><affiliation wicri:level="2"><nlm:aff id="af020">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation wicri:level="2"><nlm:aff id="af020">Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="af030">Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University, Shanghai, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University, Shanghai</wicri:regionArea><wicri:noRegion>Shanghai</wicri:noRegion></affiliation></author><author><name sortKey="Bottazzi, Maria Elena" sort="Bottazzi, Maria Elena" uniqKey="Bottazzi M" first="Maria Elena" last="Bottazzi">Maria Elena Bottazzi</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Strych, Ulrich" sort="Strych, Ulrich" uniqKey="Strych U" first="Ulrich" last="Strych">Ulrich Strych</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author><author><name sortKey="Hotez, Peter J" sort="Hotez, Peter J" uniqKey="Hotez P" first="Peter J." last="Hotez">Peter J. Hotez</name><affiliation wicri:level="1"><nlm:aff id="af010">Texas Children’s Hospital Center for Vaccine Development, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Texas Children’s Hospital Center for Vaccine Development</wicri:regionArea></affiliation><affiliation wicri:level="2"><nlm:aff id="af015">Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX</wicri:regionArea><placeName><region type="state">Texas</region></placeName></affiliation></author></analytic><series><title level="j">Vaccine</title><idno type="ISSN">0264-410X</idno><idno type="eISSN">1873-2518</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antigens, Viral (genetics)</term><term>Antigens, Viral (immunology)</term><term>CHO Cells</term><term>Coronavirus Infections (immunology)</term><term>Coronavirus Infections (prevention & control)</term><term>Cricetulus</term><term>Epitopes (chemistry)</term><term>Epitopes (immunology)</term><term>Gene Expression</term><term>Genetic Engineering</term><term>Genetic Vectors (genetics)</term><term>Immunogenicity, Vaccine</term><term>Immunoglobulin Fc Fragments (genetics)</term><term>Immunoglobulin Fc Fragments (immunology)</term><term>Mice</term><term>Middle East Respiratory Syndrome Coronavirus (immunology)</term><term>Protein Processing, Post-Translational</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (immunology)</term><term>Viral Vaccines (immunology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Antigènes viraux (génétique)</term><term>Antigènes viraux (immunologie)</term><term>Cellules CHO</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (immunologie)</term><term>Cricetulus</term><term>Expression des gènes</term><term>Fragments Fc des immunoglobulines (génétique)</term><term>Fragments Fc des immunoglobulines (immunologie)</term><term>Génie génétique</term><term>Immunogénicité des vaccins</term><term>Infections à coronavirus ()</term><term>Infections à coronavirus (immunologie)</term><term>Maturation post-traductionnelle des protéines</term><term>Protéines recombinantes (génétique)</term><term>Protéines recombinantes (immunologie)</term><term>Souris</term><term>Vaccins antiviraux (immunologie)</term><term>Vecteurs génétiques (génétique)</term><term>Épitopes ()</term><term>Épitopes (immunologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Epitopes</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antigens, Viral</term><term>Immunoglobulin Fc Fragments</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antigens, Viral</term><term>Epitopes</term><term>Immunoglobulin Fc Fragments</term><term>Recombinant Proteins</term><term>Viral Vaccines</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Genetic Vectors</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Antigènes viraux</term><term>Fragments Fc des immunoglobulines</term><term>Protéines recombinantes</term><term>Vecteurs génétiques</term></keywords><keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Antigènes viraux</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term><term>Fragments Fc des immunoglobulines</term><term>Infections à coronavirus</term><term>Protéines recombinantes</term><term>Vaccins antiviraux</term><term>Épitopes</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>Coronavirus Infections</term><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" qualifier="prevention & control" xml:lang="en"><term>Coronavirus Infections</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>CHO Cells</term><term>Cricetulus</term><term>Gene Expression</term><term>Genetic Engineering</term><term>Immunogenicity, Vaccine</term><term>Mice</term><term>Protein Processing, Post-Translational</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules CHO</term><term>Cricetulus</term><term>Expression des gènes</term><term>Génie génétique</term><term>Immunogénicité des vaccins</term><term>Infections à coronavirus</term><term>Maturation post-traductionnelle des protéines</term><term>Souris</term><term>Épitopes</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Azhar, E I" uniqKey="Azhar E">E.I. Azhar</name></author><author><name sortKey="Lanini, S" uniqKey="Lanini S">S. Lanini</name></author><author><name sortKey="Ippolito, G" uniqKey="Ippolito G">G. 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Morrison</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Malaisie</li><li>République populaire de Chine</li><li>États-Unis</li></country><region><li>Texas</li><li>État de New York</li></region></list><tree><country name="Malaisie"><noRegion><name sortKey="Nyon, Mun Peak" sort="Nyon, Mun Peak" uniqKey="Nyon M" first="Mun Peak" last="Nyon">Mun Peak Nyon</name></noRegion></country><country name="États-Unis"><noRegion><name sortKey="Nyon, Mun Peak" sort="Nyon, Mun Peak" uniqKey="Nyon M" first="Mun Peak" last="Nyon">Mun Peak Nyon</name></noRegion><name sortKey="Agrawal, Anurodh" sort="Agrawal, Anurodh" uniqKey="Agrawal A" first="Anurodh" last="Agrawal">Anurodh Agrawal</name><name sortKey="Algaissi, Abdullah" sort="Algaissi, Abdullah" uniqKey="Algaissi A" first="Abdullah" last="Algaissi">Abdullah Algaissi</name><name sortKey="Bottazzi, Maria Elena" sort="Bottazzi, Maria Elena" uniqKey="Bottazzi M" first="Maria Elena" last="Bottazzi">Maria Elena Bottazzi</name><name sortKey="Bottazzi, Maria Elena" sort="Bottazzi, Maria Elena" uniqKey="Bottazzi M" first="Maria Elena" last="Bottazzi">Maria Elena Bottazzi</name><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name><name sortKey="Hotez, Peter J" sort="Hotez, Peter J" uniqKey="Hotez P" first="Peter J." last="Hotez">Peter J. Hotez</name><name sortKey="Hotez, Peter J" sort="Hotez, Peter J" uniqKey="Hotez P" first="Peter J." last="Hotez">Peter J. Hotez</name><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><name sortKey="Naceanceno, Kevin S" sort="Naceanceno, Kevin S" uniqKey="Naceanceno K" first="Kevin S." last="Naceanceno">Kevin S. Naceanceno</name><name sortKey="Naceanceno, Kevin S" sort="Naceanceno, Kevin S" uniqKey="Naceanceno K" first="Kevin S." last="Naceanceno">Kevin S. Naceanceno</name><name sortKey="Nyon, Mun Peak" sort="Nyon, Mun Peak" uniqKey="Nyon M" first="Mun Peak" last="Nyon">Mun Peak Nyon</name><name sortKey="Peng, Bi Hung" sort="Peng, Bi Hung" uniqKey="Peng B" first="Bi-Hung" last="Peng">Bi-Hung Peng</name><name sortKey="Pollet, Jeroen" sort="Pollet, Jeroen" uniqKey="Pollet J" first="Jeroen" last="Pollet">Jeroen Pollet</name><name sortKey="Pollet, Jeroen" sort="Pollet, Jeroen" uniqKey="Pollet J" first="Jeroen" last="Pollet">Jeroen Pollet</name><name sortKey="Seid, Christopher A" sort="Seid, Christopher A" uniqKey="Seid C" first="Christopher A." last="Seid">Christopher A. Seid</name><name sortKey="Seid, Christopher A" sort="Seid, Christopher A" uniqKey="Seid C" first="Christopher A." last="Seid">Christopher A. Seid</name><name sortKey="Strych, Ulrich" sort="Strych, Ulrich" uniqKey="Strych U" first="Ulrich" last="Strych">Ulrich Strych</name><name sortKey="Strych, Ulrich" sort="Strych, Ulrich" uniqKey="Strych U" first="Ulrich" last="Strych">Ulrich Strych</name><name sortKey="Tai, Wanbo" sort="Tai, Wanbo" uniqKey="Tai W" first="Wanbo" last="Tai">Wanbo Tai</name><name sortKey="Tseng, Chien Te Kent" sort="Tseng, Chien Te Kent" uniqKey="Tseng C" first="Chien-Te Kent" last="Tseng">Chien-Te Kent Tseng</name></country><country name="République populaire de Chine"><noRegion><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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