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

• 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
• ISSN: 0264-410X; 1873-2518
• DOI: 10.1016/j.vaccine.2018.02.065

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.