Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
Identifieur interne : 001170 ( Pmc/Curation ); précédent : 001169; suivant : 001171Engineering 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.
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
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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="1"><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></affiliation></author><author><name sortKey="Agrawal, Anurodh" sort="Agrawal, Anurodh" uniqKey="Agrawal A" first="Anurodh" last="Agrawal">Anurodh Agrawal</name><affiliation wicri:level="1"><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></affiliation></author><author><name sortKey="Algaissi, Abdullah" sort="Algaissi, Abdullah" uniqKey="Algaissi A" first="Abdullah" last="Algaissi">Abdullah Algaissi</name><affiliation wicri:level="1"><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></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="1"><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></affiliation></author><author><name sortKey="Tai, Wanbo" sort="Tai, Wanbo" uniqKey="Tai W" first="Wanbo" last="Tai">Wanbo Tai</name><affiliation wicri:level="1"><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></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation wicri:level="1"><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></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></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="1"><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></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="1"><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></affiliation></author><author><name sortKey="Hotez, Peter J" sort="Hotez, Peter J" uniqKey="Hotez P" first="Peter J." last="Hotez">Peter J. 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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></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="1"><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></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="1"><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></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="1"><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></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="1"><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></affiliation></author><author><name sortKey="Agrawal, Anurodh" sort="Agrawal, Anurodh" uniqKey="Agrawal A" first="Anurodh" last="Agrawal">Anurodh Agrawal</name><affiliation wicri:level="1"><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></affiliation></author><author><name sortKey="Algaissi, Abdullah" sort="Algaissi, Abdullah" uniqKey="Algaissi A" first="Abdullah" last="Algaissi">Abdullah Algaissi</name><affiliation wicri:level="1"><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></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="1"><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></affiliation></author><author><name sortKey="Tai, Wanbo" sort="Tai, Wanbo" uniqKey="Tai W" first="Wanbo" last="Tai">Wanbo Tai</name><affiliation wicri:level="1"><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></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation wicri:level="1"><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></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></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="1"><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></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="1"><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></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="1"><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></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></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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<front><journal-meta><journal-id journal-id-type="nlm-ta">Vaccine</journal-id><journal-id journal-id-type="iso-abbrev">Vaccine</journal-id><journal-title-group><journal-title>Vaccine</journal-title></journal-title-group><issn pub-type="ppub">0264-410X</issn><issn pub-type="epub">1873-2518</issn><publisher><publisher-name>Elsevier Ltd.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29496347</article-id><article-id pub-id-type="pmc">5860679</article-id><article-id pub-id-type="publisher-id">S0264-410X(18)30252-4</article-id><article-id pub-id-type="doi">10.1016/j.vaccine.2018.02.065</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen</article-title></title-group><contrib-group><contrib contrib-type="author" id="au005"><name><surname>Nyon</surname><given-names>Mun Peak</given-names></name><xref rid="af005" ref-type="aff">a</xref><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au010"><name><surname>Du</surname><given-names>Lanying</given-names></name><xref rid="af020" ref-type="aff">d</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au015"><name><surname>Tseng</surname><given-names>Chien-Te Kent</given-names></name><xref rid="af025" ref-type="aff">e</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au020"><name><surname>Seid</surname><given-names>Christopher A.</given-names></name><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au025"><name><surname>Pollet</surname><given-names>Jeroen</given-names></name><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au030"><name><surname>Naceanceno</surname><given-names>Kevin S.</given-names></name><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au035"><name><surname>Agrawal</surname><given-names>Anurodh</given-names></name><xref rid="af025" ref-type="aff">e</xref></contrib><contrib contrib-type="author" id="au040"><name><surname>Algaissi</surname><given-names>Abdullah</given-names></name><xref rid="af025" ref-type="aff">e</xref></contrib><contrib contrib-type="author" id="au045"><name><surname>Peng</surname><given-names>Bi-Hung</given-names></name><xref rid="af025" ref-type="aff">e</xref></contrib><contrib contrib-type="author" id="au050"><name><surname>Tai</surname><given-names>Wanbo</given-names></name><xref rid="af020" ref-type="aff">d</xref></contrib><contrib contrib-type="author" id="au055"><name><surname>Jiang</surname><given-names>Shibo</given-names></name><xref rid="af020" ref-type="aff">d</xref><xref rid="af030" ref-type="aff">f</xref></contrib><contrib contrib-type="author" id="au060"><name><surname>Bottazzi</surname><given-names>Maria Elena</given-names></name><email>bottazzi@bcm.edu</email><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib><contrib contrib-type="author" id="au065"><name><surname>Strych</surname><given-names>Ulrich</given-names></name><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au070"><name><surname>Hotez</surname><given-names>Peter J.</given-names></name><xref rid="af010" ref-type="aff">b</xref><xref rid="af015" ref-type="aff">c</xref></contrib></contrib-group><aff id="af005"><label>a</label>Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur, Malaysia</aff><aff id="af010"><label>b</label>Texas Children’s Hospital Center for Vaccine Development, USA</aff><aff id="af015"><label>c</label>Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA</aff><aff id="af020"><label>d</label>Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA</aff><aff id="af025"><label>e</label>Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX, USA</aff><aff id="af030"><label>f</label>Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University, Shanghai, China</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author at: Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA. <email>bottazzi@bcm.edu</email></corresp><fn id="fn1"><label>1</label><p id="np030">M.P.N., L.D. and C.-T.K.T. contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>26</day><month>2</month><year>2018</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>27</day><month>3</month><year>2018</year></pub-date><pub-date pub-type="epub"><day>26</day><month>2</month><year>2018</year></pub-date><volume>36</volume><issue>14</issue><fpage>1853</fpage><lpage>1862</lpage><history><date date-type="received"><day>12</day><month>10</month><year>2017</year></date><date date-type="rev-recd"><day>12</day><month>2</month><year>2018</year></date><date date-type="accepted"><day>16</day><month>2</month><year>2018</year></date></history><permissions><copyright-statement>© 2018 Elsevier Ltd. All rights reserved.</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>Elsevier Ltd</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 id="ab005"><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></abstract><kwd-group id="kg005"><title>Keywords</title><kwd>Middle East respiratory syndrome coronavirus</kwd><kwd>Receptor binding domain</kwd><kwd>Chinese hamster ovary cells</kwd></kwd-group></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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