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

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.

Keywords: Chinese hamster ovary cells; Middle East respiratory syndrome coronavirus; Receptor binding domain.

Fig. 1

Expression of MERS S377-588-Fc in adCHO cells. (a) Map of pOpti_S377-588-Fc. (b) Chromatogram of Protein A purification of MERS S377-588 from culture media of adCHO cells. Shown is the UV absorption at 280 nm (blue line) during elution with a pH gradient (purple line). Two peaks were eluted, followed by Western blotting and SDS-PAGE gel analysis. (c) Western blotting of denatured SDS-PAGE using Rb-Anti-MERS RBD antibodies. (d) Non-denatured SDS-PAGE. (e) Western blotting of non-denatured SDS-PAGE using Rb-anti-Bovine (Fab)2 antibodies.

Fig. 2

Selection of signal peptides and gradual stepwise increase of MTX concentration for DNA amplification. (a) Comparison of relative productivity of linearized constructs with different signal peptides derived from interleukin 2 (IL2), IgK light chain (IgK), human serum albumin (SA) and azurocidin (Azu). (b) Area of Peak 1 (IgG from FBS, red area) and Peak 2 (S377-588-Fc, blue area) were measured to estimate relative productivity. (c) Relative productivity of S377-588-Fc increased in the presence of MTX.

Fig. 3

Biophysical characterization and PTM analysis of MERS S377-588-Fc protein. (a) CD spectrum. (b) Melting curve. (c) Comparison of S377-588-Fc expressed in adCHO and HEK293T cells. (d) PTM analysis using PNGaseF, O-glycosidase and neuraminidase (1: control, 2: PNGaseF, 3: O-glycosidase, 4: neuraminidase, lane 5: O-glycosidase + neuraminidase, 6: PNGaseF + O-glycosidase + neuraminidase).

Fig. 4

Functionality and antigenicity of MERS S377-588-Fc proteins. CHO-#1 and CHO-#3 represent two different batches of the S377-588-Fc proteins purified from adCHO cells, and HEK indicates RBD protein purified from HEK293T. (a) Co-IP analysis of interactions between S377-588-Fc expressed from adCHO and HEK293T. The interactions between S377-588-Fc and hDPP4 in the Huh-7 cell lysates were detected by Co-IP analysis, using anti-hDPP4 and anti-MERS-RBD antibodies. Anti-SARS-RBD: SARS-CoV RBD-specific mAb 33G4. (b) Quantification of the interaction between S377-588-Fc (40 μg/mL) and hDPP4 by flow cytometry analysis in hDPP4-expressing Huh-7 cells. MFI: median fluorescence intensity. For (a) and (b), a HEK293T-expressed SARS-CoV RBD protein (e.g., SARS RBD-Fc) was used as a control, and this protein did not interact with hDPP4. (c) ELISA was performed to detect the binding between MERS S377-588-Fc (2 μg/mL) and recombinant hDPP4. Binding affinity was characterized as OD450 value. (d) Similar to S377-588-Fc expressed in HEK293T cells, S377-588-Fc proteins expressed in adCHO cells maintained good antigenicity in binding MERS-CoV RBD-specific neutralizing antibodies (including Mersmab1, m336, m337, and m338). (e) ELISA was performed to compare the binding between the above neutralizing mAbs and denatured (with DTT) or non-denatured (no DTT) MERS S377-588-Fc proteins expressed in both adCHO and HEK293T. ^***, P < 0.001. Control in (c)–(e): human IgG Fc (hFc) protein. Error bars indicate standard error.

Fig. 5

Immunogenicity of MERS S377-588-Fc protein expressed in adCHO cells. Sera from RBD-immunized hDPP4-Tg mice were tested by ELISA for RBD-specific IgG antibodies. Plates were coated with S377-588-Fc (1 μg/mL), and treated with or without DTT, followed by incubation with serially diluted mouse sera (n = 4). Error bars indicate standard error. ^***, P < 0.001.

Fig. 6

Growth curve comparison of heterogeneous susCHO cell pools and clone 1B11. The high-producing clone 1B11 has a lower growth rates than the heterogeneous non-clonal cell pools.