A recombinant receptor-binding domain of MERS-CoV in trimeric form protects human dipeptidyl peptidase 4 (hDPP4) transgenic mice from MERS-CoV infection

Abstract

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) was first identified in 2012, and it continues to threaten human health worldwide. No MERS vaccines are licensed for human use, reinforcing the urgency to develop safe and efficacious vaccines to prevent MERS. MERS-CoV spike protein forms a trimer, and its receptor-binding domain (RBD) serves as a vaccine target. Nevertheless, the protective efficacy of RBD in its native trimeric form has never been evaluated. In this study, a trimeric protein, RBD-Fd, was generated by fusing RBD with foldon trimerization motif. It bound strongly to the receptor of MERS-CoV, dipeptidyl peptidase 4 (DPP4), and elicited robust RBD-specific neutralizing antibodies in mice, maintaining long-term neutralizing activity against MERS-CoV infection. RBD-Fd potently protected hDPP4 transgenic mice from lethal MERS-CoV challenge. These results suggest that MERS-CoV RBD in its trimeric form maintains native conformation and induces protective neutralizing antibodies, making it a candidate for further therapeutic development.

Keywords: Foldon trimerization motif; MERS; MERS-CoV; Neutralization; Protection; Receptor-binding domain; Spike protein; hDPP4-transgenic mice.

Graphical abstract

Fig. 1

Construction, characterization and antigenicity evaluation of MERS-CoV RBD-Fd protein. (A) Schematic structure of MERS-CoV S1 subunit and construction of RBD-Fd. A His₆ tag was added at the C-terminus of RBD-Fd for easy purification. (B) SDS-PAGE and Western blot analysis of purified RBD-Fd protein. Denatured (boiled) or non-denatured (non-boiled) samples (5 µg) were subjected to SDS-PAGE (left) and Western blot analysis (right) using MERS-CoV S1-specific antibody. The molecular weight marker (kDa) is indicated on the left. (C) SEC-MALS analysis of purified of RBD-Fd protein. RBD-Fd (2 mg/ml, 100 μl) was subjected to the analysis. The data are presented as mean molar mass (Da). (D) ELISA detection of the binding between RBD-Fd and RBD-specific neutralizing mAbs. RBD-Fd (2 μg/ml)-coated ELISA plates were incubated with serially diluted mAbs for the test.

Fig. 2

Functionality of RBD-Fd protein in binding to the DPP4 receptor of MERS-CoV. (A-B) Co-IP and Western blot analysis of the binding between RBD-Fd and cell-associated DPP4. RBD-Fd was incubated with DPP4-expressing Huh-7 cell lysates in the presence of Ni-NTA Superflow, followed by detection of the binding by Western blot using DPP4- (0.2 µg/ml) (A) or MERS-CoV RBD-specific antibody (B) (1:5000). sDPP4 and RBD-Fd proteins only were included as the controls. The molecular weight marker (kDa) is indicated on the left. (C) ELISA measurement of the binding between RBD-Fd and sDPP4 proteins. RBD-Fd (2 μg/ml)-coated ELISA plates were incubated with serially diluted sDPP4 for the test.

Fig. 3

Inhibitory activity of RBD-Fd and its immunogenicity and neutralizing ability in immunized mice. (A) Inhibitory activity of RBD-Fd protein against infections of pseudotyped MERS-CoV (EMC2012) in Huh-7 cells. An unrelated SARS-CoV RBD protein was included as the control. The data are presented as 50% inhibition dose (ID₅₀). (B) MERS-CoV S1-specific IgG antibody titers. Mouse sera from different months were used for the test. MERS-CoV S1-specific IgG1 (C) and IgG2a (D) antibody titers. Mouse sera from 2 and 6 months post-1st immunization were used for the test. The antibody titers are expressed as the endpoint dilution that remains positively detectable, and presented as mean titers ±SD of five mice in each group. RBD-Fd-induced neutralizing antibodies against infections of pseudotyped (E) and live (F) MERS-CoV of EMC2012 strain, as well as pseudotyped MERS-CoV of representative strains isolated from the 2012 (L506F), 2013 (A434V, A431P-A482V, S460F), 2014 (Q522H, T424I), and 2015 (V530L, V534A) outbreaks, respectively (G). Shown here are respective mutations in the RBD of S protein, as compared with those of EMC2012 strain (Qiu et al., 2016). Neutralizing antibody titers were calculated as the reciprocal of the highest dilution of sera that resulted in a complete inhibition of MERS pseudovirus infection (E and G) or MERS-CoV-induced CPE (F) in at least 50% of the wells (NT₅₀). Mouse sera from 2 and 6 months were used for the test, and the data are presented as mean titers ±SD of five mice in each group. For (B)-(G), PBS was included as the control.

Fig. 4

Protective efficacy of RBD-Fd in hDPP4-Tg mice. Groups of 6 mice were challenged with MERS-CoV at 12 weeks post-last dose and then observed for 3 weeks for survival rate (A) and body weight changes (C). The data are presented as mean with SD. (B) Neutralizing antibodies were detected in the sera of immunized mice before MERS-CoV challenge. Neutralizing antibody titers are presented as mean NT₅₀ ±SD of six mice in each group. For (A)–(C), PBS was included as the control. (D) Evaluation of pathological changes in lung tissues of challenged mice. Representative images from mice immunized with RBD-Fd (a) and PBS (c) challenged with MERS-CoV (EMC2012) and normal mice (b) are shown. Lung tissue sections were stained with hematoxylin and eosin (H&E) and observed under light microscopy (100×magnification).