Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

Mun Peak Nyon, Lanying Du, Chien-Te Tseng, Christopher A. Seid, Jeroen Pollet, Kevin S. Naceanceno, Anurodh Agrawal, Abdullah Algaissi, Bihung Peng, Wanbo Tai, Shibo Jiang, Maria Elena Bottazzi, Ulrich Strych, Peter J. Hotez

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
JournalVaccine
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Coronavirus
Coronavirinae
Chinese hamsters
Cricetulus
Ovary
engineering
Immunoglobulin Fc Fragments
Vaccines
cell lines
vaccines
antigens
Antigens
Cell Line
Recombinant Proteins
recombinant proteins
Immunoglobulin G
Neutralizing Antibodies
neutralizing antibodies
Coronavirus Spike Glycoproteins
manufacturing

Keywords

  • Chinese hamster ovary cells
  • Middle East respiratory syndrome coronavirus
  • Receptor binding domain

ASJC Scopus subject areas

  • Molecular Medicine
  • Immunology and Microbiology(all)
  • veterinary(all)
  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

Cite this

Nyon, M. P., Du, L., Tseng, C-T., Seid, C. A., Pollet, J., Naceanceno, K. S., ... Hotez, P. J. (Accepted/In press). Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen. Vaccine. https://doi.org/10.1016/j.vaccine.2018.02.065

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen. / Nyon, Mun Peak; Du, Lanying; Tseng, Chien-Te; Seid, Christopher A.; Pollet, Jeroen; Naceanceno, Kevin S.; Agrawal, Anurodh; Algaissi, Abdullah; Peng, Bihung; Tai, Wanbo; Jiang, Shibo; Bottazzi, Maria Elena; Strych, Ulrich; Hotez, Peter J.

In: Vaccine, 01.01.2018.

Research output: Contribution to journalArticle

Nyon, MP, Du, L, Tseng, C-T, Seid, CA, Pollet, J, Naceanceno, KS, Agrawal, A, Algaissi, A, Peng, B, Tai, W, Jiang, S, Bottazzi, ME, Strych, U & Hotez, PJ 2018, 'Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen', Vaccine. https://doi.org/10.1016/j.vaccine.2018.02.065
Nyon, Mun Peak ; Du, Lanying ; Tseng, Chien-Te ; Seid, Christopher A. ; Pollet, Jeroen ; Naceanceno, Kevin S. ; Agrawal, Anurodh ; Algaissi, Abdullah ; Peng, Bihung ; Tai, Wanbo ; Jiang, Shibo ; Bottazzi, Maria Elena ; Strych, Ulrich ; Hotez, Peter J. / Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen. In: Vaccine. 2018.
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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.",
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AU - Pollet, Jeroen

AU - Naceanceno, Kevin S.

AU - Agrawal, Anurodh

AU - Algaissi, Abdullah

AU - Peng, Bihung

AU - Tai, Wanbo

AU - Jiang, Shibo

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AU - Hotez, Peter J.

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AB - 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.

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