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

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...

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Bibliographic Details
Published in:Vaccine 2018-03, Vol.36 (14), p.1853-1862
Main Authors: Nyon, Mun Peak, Du, Lanying, Tseng, Chien-Te Kent, Seid, Christopher A., Pollet, Jeroen, Naceanceno, Kevin S., Agrawal, Anurodh, Algaissi, Abdullah, Peng, Bi-Hung, Tai, Wanbo, Jiang, Shibo, Bottazzi, Maria Elena, Strych, Ulrich, Hotez, Peter J.
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antibody response
Antigens
Antigens, Viral - genetics
Antigens, Viral - immunology
Binding
Chinese hamster ovary cells
CHO Cells
Cloning
Coronaviridae
Coronavirus Infections - immunology
Coronavirus Infections - prevention & control
Coronaviruses
Cricetulus
Deoxyribonucleic acid
Dihydrofolate reductase
Dipeptidyl-peptidase IV
DNA
Enzymes
Epitopes - chemistry
Epitopes - immunology
Fc receptors
Gene amplification
Gene Expression
Genetic Engineering
Genetic Vectors - genetics
Immunogenicity, Vaccine
Immunoglobulin Fc Fragments - genetics
Immunoglobulin Fc Fragments - immunology
Immunoglobulin G
Immunoglobulins
Immunology
Interleukin 2
Leukocytes (eosinophilic)
Methotrexate
Mice
Middle East respiratory syndrome coronavirus
Middle East Respiratory Syndrome Coronavirus - immunology
Neutralizing
Peptidase
Peptides
Plasmids
Protein Processing, Post-Translational
Proteins
Receptor binding domain
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Respiratory diseases
Rodents
Spike protein
Toxicity
Transgenic mice
Vaccines
Viral Vaccines - immunology
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Summary: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.
ISSN:0264-410X
1873-2518
DOI:10.1016/j.vaccine.2018.02.065