Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development

Abstract

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine. In 2003, Farzan and colleagues successfully identified the receptor of SARS-CoV, angiotensin-converting enzyme 2 (ACE2) (7), and a 193-amino-acid fragment in the spike (S) protein (residues 318 to 510) as the receptor-binding domain (RBD) (8). We found that SARS-CoV S-RBD contains a critical neutralizing site (9) which induces potent neutralizing antibodies and protection against SARS-CoV infection in an animal model (10).Since MERS-CoV is genetically related to SARS-CoV (1), we compared their S protein sequences and predicted that the RBD of MERS-CoV might be located in the region spanning residues 377 to 662 of the S1 subunit (Fig. 1). Using the Swiss-Model Workplace homology modeling server (11) and basing our work on the X-ray crystal structure of the SARS-CoV S-RBD (Protein Data Bank [PDB] identification no. 2DD8) (12), we predicted the conformational structure of the region consisting of residues 377 to 662 in the S1 subunit of the MERS-CoV S protein (13). We noticed that the SARS-CoV S-RBD and the predicted MERS-CoV S-RBD possessed similar core structures but had an extended secondary structure consisting predominantly of the receptor-binding motifs (RBM) (12,14). The extended region in MERS-CoV S-RBD is much longer than that in SARS-CoV S-RBD, suggesting that MERS-CoV and SARS-CoV use different receptors. Indeed, it has been proven that dipeptidyl peptidase-4 (DPP4; also known as CD26) is the functional receptor of MERS-CoV (15).We then constructed MERS-CoV S-RBD based on the synthesized codon-optimized MERS-CoV S sequences (GenBank accession no. AFS88936.1) and fused it to Fc of human IgG using pFUSE-hIgG1-Fc2 expression vector (here named Fc) (InvivoGen, San Diego, CA). The SARS-CoV S-RBD-Fc was constructed by fusing RBD of codon-optimized SARS-CoV S sequence into the Fc vector referred to above as a control (Fig. 1) (16). The S-RBD-Fc proteins were expressed in 293T cell culture supernatant and purified by protein A affinity chromatography (GE Healthcare, Piscataway, NJ) (17). We found that both MERS-CoV and SARS-CoV S-RBD-Fc proteins were highly purified from transfected culture supernatants ( Fig. 2A, panel a). MERS-CoV S-RBD-Fc could be recognized by an MERS-CoV S-specific polyclonal antibody (1:1,000), while SARS-CoV S-RBD-Fc could not react with this antibody, as detected by Western blotting (Fig. 2A, panel b).Using analysis performed by Western blotting, we found that DPP4 was highly express...

Crystal Structure of the Receptor-Binding Domain from Newly Emerged Middle East Respiratory Syndrome Coronavirus

“…During the submission and review of the present study, two other studies independently mapped the MERS-CoV RBD fragments (residues 358 to 588 and 377 to 662), both of which are similar to the one identified in the present study (residues 367 to 588) (42,43). These studies also showed that the RBD efficiently elicits neutralizing antibodies, confirming that the crystal structure of MERS-CoV can be useful in structure-based vaccine design.…”

MERS – A cautionary tale

“…Based on their previous experience in developing RBD-based SARS vaccines [16][17][18], the researchers have also designed a subunit MERS vaccine candidate consisting of an optimized fragment of MERS-CoV S protein RBD and a human IgG Fc, S377-588-Fc [19,20]. This vaccine candidate elicited highly potent neutralizing antibody responses, which protected hDPP4-transduced mice from MERS-CoV challenge, suggesting promise for its further development as an effective and safe MERS vaccine [21].…”

Current advancements and potential strategies in the development of MERS-CoV vaccines

“…The induction of neutralizing activity by a recombinant protein fragment containing residues 377–662 of MERS-CoV RBD has also been demonstrated. Particularly, a truncated 212-amino acid RBD fragment containing residues 377–588 (inside RBD: residues 367–606) of MERS-CoV S protein induced strong MERS-CoV S-specific antibodies in vaccinated mice, blocking the binding of MERS-CoV RBD to viral receptor DPP4 and effectively neutralizing MERS-CoV infection, thus providing promise for further development as an effective and safe MERS vaccine [56–57]. …”