Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen Mun Peak Nyon a,b,c,1, Lanying Du d,1, Chien-Te Kent Tseng e,1, Christopher A. Seid b,c, Jeroen Pollet b,c, Kevin S. Naceanceno b,c, Anurodh Agrawal e, Abdullah Algaissi e, Bi-Hung Peng e, Wanbo Tai d, Shibo Jiang d,f, Maria Elena Bottazzi b,c,⇑, Ulrich Strych b,c, Peter J. Hotez b,c a Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Kuala Lumpur, Malaysia b Texas Children’s Hospital Center for Vaccine Development, USA c Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA d Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USA e Department of Microbiology and Immunology & Center of Biodefense and Emerging Diseases, University of Texas Medical Branch, Galveston, TX, USA f Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University, Shanghai, China a r t i c l e i n f o Article history: Received 12 October 2017 Received in revised form 12 February 2018 Accepted 16 February 2018 Available online xxxx Keywords: Middle East respiratory syndrome coronavirus Receptor binding domain Chinese hamster ovary cells a b s t r a c t 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 lM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding speci- ficity 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 pre- pare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield mono- clonal suspension CHO cell line. � 2018 Published by Elsevier Ltd. 1. Introduction With over 712 deaths and 2040 confirmed cases since its orig- inal appearance on the Arabian peninsula in 2012 [1], Middle East Respiratory syndrome (MERS) coronavirus (MERS-CoV) has emerged as an important global pathogen and potential pandemic threat. There remains a critical need for a vaccine targeting MERS-CoV [2], and the newly established Coalition for Epidemic Preparedness Innovation (CEPI) has now designated research and development for the MERS-CoV vaccine as a global priority [3]. Recently, Phase I studies of DNA-based vaccines against MERS-CoV showed that 98% of vaccinated volunteers generated antibody against MERS CoV [4]. However, to date there is no licensed DNA vaccine for humans due in part to questions about their long-term safety, and their ability to induce high titers of protective or neutralizing antibodies relative to recombinant protein-based vaccines [5,6]. A lead candidate for such a protein-based vaccine is the receptor-binding domain (RBD) of the MERS-CoV spike (S) protein. The MERS-CoV RBD plays an essential role in host receptor binding, https://doi.org/10.1016/j.vaccine.2018.02.065 0264-410X/� 2018 Published by Elsevier Ltd. ⇑ Corresponding author at: Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA. E-mail address: