Antibodies induced by receptor-binding domain in spike protein of SARS-CoV do not cross-neutralize the novel human coronavirus hCoV-EMC

Dear Editor,

Most recently, Chan et al.¹ reported, in this journal, that convalescent SARS patients' sera may contain cross-reactive antibodies against the emerging novel human coronavirus EMC/2012 (hCoV-EMC) detected by both immunofluorescent and neutralizing antibody tests.¹

SARS coronavirus (SARS-CoV), the causative agent of SARS, uses the angiotensin-converting enzyme 2 (ACE2) as its cellular receptor to bind to the target cells,² and a 193-amino acid fragment (residues 318–510) in the S1 subunit of vial spike (S) protein is the identified receptor-binding domain (RBD).³ The recent emergence of hCoV-EMC has caused 17 people infected including 11 deaths (http://www.who.int/csr/don/2013_03_26/en/), raising serious concern about its potential pandemic. Unlike SARS-CoV, hCoV-EMC uses a different receptor, dipeptidyl peptidase-4 (DPP4),⁴ for its binding and entry into the target cell. We have predicted that a 286-amino acid fragment (residues 377–662) of hCoV-EMC S1 region contains the viral RBD.⁵

Previously we reported that the RBD of SARS-CoV S protein contains multiple neutralizing epitopes that induce potent neutralizing antibodies and protection against SARS-CoV infection in animal models.6, 7, 8 Thus, neutralizing antibodies targeting the S protein, particularly the RBD, play the most important roles in the inhibition of viral infection. Since both SARS-CoV and hCoV-EMC genetically belong to the genus betacoronavirus,9, 10 we thus speculate that the antibodies induced by the RBD of SARS-CoV may have cross-reactivity or cross-neutralizing activity against hCoV-EMC.

To prove this, we first tested the reactivity of a series of SARS-CoV RBD-specific monoclonal antibodies (mAbs)6, 11 with recombinant proteins containing S1 (residues 18–725) and putative RBD (residues 377–662) in S of hCoV-EMC. We found that all of these mAbs that can recognize the conformational (Conf I–VI, Group A–E) or linear epitopes in RBD of SARS-CoV had low to no binding (A450 < 0.3) to the RBD and S1 proteins of hCoV-EMC at the concentration as high as 10 μg/ml, while they had a strong binding to a recombinant RBD protein of SARS-CoV at the tested concentration of 1 μg/ml (Fig. 1 A).⁷ These results suggest that the antibodies induced by the RBD of SARS-CoV S protein did not cross-react with the RBD and S1 protein of hCoV-EMC.

Figure 1.

Cross-reactivity and cross-neutralization activity of SARS-CoV S-RBD-specific antibodies against hCoV-EMC. (A) Reactivity of SARS-CoV S-RBD-specific mAbs with RBD and/or S1 protein of hCoV-EMC and SARS-CoV as detected by ELISA. Conf I–VI, Group A–E, and linear mAbs represent the mAbs targeting the conformational and linear epitopes in RBD of SARS-CoV S protein, respectively. HA-7 mAb specific to hemagglutinin (HA) of H5N1 influenza virus was used as the negative control. The data are presented as mean A450 ± standard deviation (SD) of duplicate wells. Neutralization of SARS-CoV S-RBD-specific mAbs (B) and SARS-CoV S-RBD protein-vaccinated mouse antisera (C) against hCoV-EMC and SARS-CoV infection by pseudovirus neutralization assay. The data are presented as mean percentages of neutralization ± SD of duplicate wells.

We next detected the neutralizing activity of the representative SARS-CoV S-RBD-specific neutralizing mAbs against hCoV-EMC infection in Huh-7 cells that express DPP4 receptor for hCoV-EMC⁴ and against SARS-CoV infection in ACE2/293T cells expressing the receptor for SARS-CoV,⁷ using our established pseudovirus neutralization assay. As shown in Fig. 1B, in an exception of the mAb 24H8 (Conf I) that had a lower neutralization, all other mAbs including 27C1, 18D9, 35B5, 33G4, 45F6, and S38, which recognize conformational epitopes Conf II–VI and Group B of RBD of SARS-CoV,6, 11 had >90% and ≥70% neutralization of SARS-CoV pseudovirus at the concentration of 10 and 1 μg/ml, respectively. However, all these mAbs could not neutralize hCoV-EMC pseudovirus at the concentration as high as 10 μg/ml, suggesting that the SARS-CoV RBD-specific neutralizing mAbs had low to no cross-neutralization against hCoV-EMC.

To further confirm our conclusion, we performed another experiment to test the neutralizing activity of antibodies in the sera of SARS-CoV S-RBD protein-vaccinated mice. As shown in Fig. 1C, none of the tested sera neutralized hCoV-EMC pseudovirus at the dilution of 1:10, while they could potently neutralize SARS-CoV pseudovirus infection in ACE2/293T cells at the dilution of 1:10,240. These results confirm that the antibodies induced by the RBD of SARS-CoV S1 protein cannot cross-neutralize hCoV-EMC infection. Therefore, the epitopes in SARS-CoV S protein that elicit the antibodies with cross-reactivity and cross-neutralizing activity against hCoV-EMC may not be located in the RBD in S1 subunit of SARS-CoV.

By bioinformatic analysis of S proteins of SARS-CoV and hCoV-EMC, Chan et al.¹ found that an immunogenic region hCoV-EMC S (emc-II) and that in SARS-CoV S (sars-I) overlapped the heptad repeat 2 (HR2) region of the S2 domain of both hCoV-EMC and SARS-CoV, while SARS-CoV S-HR2 harbors an epitope for broadly neutralizing antibody in the case of SARS-CoV.¹² They thus believed that the epitope located in this region may be responsible for inducing cross-neutralizing antibodies against both hCoV-EMC and SARS-CoV. However, an experiment to prove this hypothesis is warranted.

Potentials conflicts of interest

No reported conflicts.