Receptor-binding domain-based subunit vaccines against MERS-CoV

Abstract

Development of effective vaccines, in particular, subunit-based vaccines, against emerging Middle East respiratory syndrome (MERS) caused by the MERS coronavirus (MERS-CoV) will provide the safest means of preventing the continuous spread of MERS in humans and camels. This review briefly describes the structure of the MERS-CoV spike (S) protein and its receptor-binding domain (RBD), discusses the current status of MERS vaccine development and illustrates the strategies used to develop RBD-based subunit vaccines against MERS. It also summarizes currently available animal models for MERS-CoV and proposes a future direction for MERS vaccines. Taken together, this review will assist researchers working to develop effective and safe subunit vaccines against MERS-CoV and any other emerging coronaviruses that might cause future pandemics.

Keywords: MERS-CoV; Middle East respiratory syndrome coronavirus; Receptor-binding domain; Subunit vaccines.

Fig. 1

Schematic structures of MERS-CoV and its spike protein RBD. (A) Schematic structure of MERS-CoV. MERS-CoV contains a positive, single-stranded RNA and four structural proteins, including S, M, E and N. (B) Spike protein of MERS-CoV and its RBD. MERS-CoV S protein contains S1 and S2 subunits, and their functional regions with specific amino acid residues are shown. SP, signal peptide. RBD, receptor-binding domain. RBM, receptor-binding motif within RBD. FP, fusion peptide. HR1 and HR2, heptad repeats 1 and 2. TM, transmembrane domain. CP, cytoplasmic tail.

Fig. 2

Crystal structures of MERS-CoV and SARS-CoV RBDs and their complexes with respective receptors. (A) Crystal structure of MERS-CoV RBD. The receptor binding motif (RBM) is in magenta. Critical residues in the RBD-DPP4 binding interface are shown in cyan (PDB ID: 4KQZ). The residues are selected based on their direct and strong interactions with receptor residues. (B) Crystal structure of MERS-CoV RBD (blue) complexed with its receptor human DPP4 (lemon) (PDB ID: 4KR0). (C) Crystal structure of SARS-CoV RBD. The receptor binding motif (RBM) is in magenta. Critical residues in the RBD-ACE2 binding interface are shown in cyan (PDB ID: 2GHW). The residues are selected based on their direct and strong interactions with receptor residues. (D) Crystal structure of SARS-CoV RBD (blue) complexed with its receptor human ACE2 (lemon) (PDB ID: 2AJF). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Characterization of MERS-CoV S377-662-Fc protein for its antigenicity and receptor binding affinity, and comparison of its immunogenicity via s.c. and i.n. routes. (A) SDS-PAGE (left) and Western blot (right) detection of S377-662-Fc protein. Samples (5 μg), either boiled (denatured) or nonboiled (nondenatured), were subjected to SDS-PAGE, followed by Coomassie Blue staining or Western blot, using anti-MERS-CoV-S1 antibodies (1: 1000) developed in our laboratories. The protein molecular weight marker (kDa) is indicated on the left. (B) Co-immunoprecipitation and Western blot analysis of S377-662-Fc binding sDPP4, the receptor for MERS-CoV (left), and cell-associated DPP4 in Huh-7 cells (right). The binding affinity was tested using anti-human DPP4 monoclonal antibody (1 μg/ml, R&D Systems, Minneapolis, MN) and anti-MERS-CoV-S1 polyclonal antibodies (1: 1000). (C) Comparison of long-term humoral immune responses induced by S377-662-Fc protein in s.c.- and i.n.-immunized mice. The mouse sera were collected at 0, 1, 2, 3, 4 and 6 months and 10 days post-last boost, and the data are presented as mean (IgG endpoint titers) ± standard deviation (SD) of five mice per group. (D) Comparison of mucosal immune responses induced by S377-662-Fc protein in s.c.- and i.n.-immunized mice. The mouse lung washes were collected at 10 days post-last boost, and the data are presented as mean (IgA A450) ± SD of five mice per group.^* indicates significant differences between S377-662-Fc i.n. group and the other groups (P < 0.05). Comparison of neutralizing antibody response against MERS-CoV infection in Vero E6 cells in sera (E) and lung washes (F) of mice s.c.- and i.n.-vaccinated with S377-662-Fc protein. Neutralizing antibody titers are expressed as the reciprocal of the highest dilutions of samples that completely inhibit virus-induced cytopathic effect (CPE) in at least 50% of the wells (NT₅₀). Samples were collected at 10 days post-last boost, and the data are presented as mean (neutralizing antibody titers) ± SD of five mice per group.^* indicates significant differences between the S377-662-Fc i.n. group and the other groups (P < 0.05). For C–F, mice injected with PBS plus respective adjuvants (Montanide ISA51 for s.c. and Poly (I:C) for i.n.) were included as the control.

Fig. 4

Comparison of four MERS-CoV RBD protein fragments for their antigenicity, receptor binding affinity, immunogenicity and neutralizing potential. (A) Construction of S377-588-Fc, S358-588-Fc, S367-588-Fc and S367-606-Fc by fusing RBD fragments containing corresponding residues of MERS-CoV S protein with Fc of human IgG. (B) Comparison of binding of RBD fragments with MERS-CoV RBD-specific monoclonal antibody Mersmab1 by ELISA. The data are presented as mean (IgG A450) ± SD of duplicate wells. (C) Comparison of receptor binding affinity of RBD fragments with sDPP4 by ELISA. The binding shows dose dependency. The data are presented as mean (IgG A450) ± SD of duplicate wells. (D) Comparison of immunogenicity of RBD fragments in immunized mice. Mouse sera were collected at 10 days post-3rd vaccination, and the data are presented as mean (IgG endpoint titers) ± SD of five mice per group.^* indicates significant differences between S377-588-Fc and S367-588-Fc with other groups (P < 0.05). (E) Comparison of neutralizing antibody response against MERS-CoV infection in Vero E6 cells in RBD fragment-immunized mice. Neutralizing antibody titers are expressed as the reciprocal of the highest dilutions of sera that completely inhibit virus-induced CPE in at least 50% of the wells (NT₅₀). Mouse sera were collected at 10 days post-3rd vaccination, and the data are presented as mean (neutralizing antibody titers) ± SD of five mice per group.^* indicates significant differences between S377-588-Fc and other groups (P < 0.05).