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.

Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development

“…Using CHO cell technology, we have recently obtained the high-yield producer stable cell clones for production of glycan-masking Pan-H5 vaccines [29]. Similar strategies have been also reported for production of a homogeneous HIV-1 envelope SOSIP trimer vaccine [30] and a MERS-coronavirus vaccine antigen [31].…”

“…Cell clones that survived following treatment with 1 mM MTX were collected and analyzed by Western blotting using anti-rH7 antibodies (GeneTex Inc.) to confirm CHO-rH7 expression. The final selected rH7-expressing clones were cultured, and the culture supernatants were harvested for rH7 purification using nickel-chelated resin affinity chromatography (Tosoh), as previously described [29][30][31]. The purified rH7 proteins were treated with endoglycosidase H (Endo H) (New England BioLabs) or N-glycosidase F (PNGase F) (New England BioLabs) for Western blotting characterization.…”

Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses

“…However, after comparing several versions of MERS-CoV RBD fragments with different lengths, it was found that a truncated RBD (residues 377-588) had the highest DPP4binding affinity and induced the highest-titer IgG antibodies and neutralizing antibodies against MERS-CoV, identifying its role as a critical neutralizing domain . Subsequently, several MERS-CoV subunit vaccines have been designed based on the identified critical neutralizing domain of RBD fragment, including those expressed in a stable CHO cell line (S377-588-Fc), fusing with a trimeric motif foldon (RBD-Fd), or containing single or multiple mutations in the RBD of representative human and camel strains from the 2012-2015 MERS outbreaks (Tai et al, 2016Nyon et al, 2018). These RBD proteins maintain good conformation, functionality, antigenicity, and immunogenicity, with ability to bind the DPP4 receptor and RBD-specific neutralizing mAbs and to elicit robust neutralizing antibodies cross-neutralizing multiple strains of MERS pseudoviruses and live MERS-CoV (Tai et al, 2016Nyon et al, 2018).…”

MERS coronavirus outbreak: Implications for emerging viral infections

“…Use of an adjuvant, particularly MF59 or AddaVax, significantly improved both the humoral and T cell responses in subcutaneously immunized mice (Zhang et al, 2016b). Recently, a high-yield CHO cell line capable of large-scale production of this S1 RBD-Fc fusion product was described, strengthening the possibility of sustainable manufacture and human testing for this potential vaccine antigen (Nyon et al, 2018). Another recent study showed that 5 recombinant RBDs incorporating mutations which arose in different MERS-CoV outbreaks or in camel strains could induce neutralizing antibody responses against several MERS-CoV pseudoviruses (Tai et al, 2017).…”

“…In vivo (Mouse) Humoral response in mice; potential intranasal administration; improved by adjuvant; divergent strains/ escape mutants; CHO cell line (Du et al, 2013;Ma et al, 2014;Nyon et al, 2018;Tai et al, 2017;Zhang et al, 2015Zhang et al, , 2016b T cell and neutralizing antibody responses; entering human clinical trials; potential for veterinary use- (Langenmayer et al, 2018;Volz et al, 2015) ad5 or ad41 adenovirus expressing full-length S…”