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. 2016 Dec 16;91(1):e01651-16.
doi: 10.1128/JVI.01651-16. Print 2017 Jan 1.

Recombinant Receptor-Binding Domains of Multiple Middle East Respiratory Syndrome Coronaviruses (MERS-CoVs) Induce Cross-Neutralizing Antibodies against Divergent Human and Camel MERS-CoVs and Antibody Escape Mutants

Wanbo Tai  1   2 , Yufei Wang  1   2 , Craig A Fett  3 , Guangyu Zhao  2 , Fang Li  4 , Stanley Perlman  3 , Shibo Jiang  1   5 , Yusen Zhou  6 , Lanying Du  7
Affiliations

Recombinant Receptor-Binding Domains of Multiple Middle East Respiratory Syndrome Coronaviruses (MERS-CoVs) Induce Cross-Neutralizing Antibodies against Divergent Human and Camel MERS-CoVs and Antibody Escape Mutants

Wanbo Tai et al. J Virol. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains.

Importance: MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity, and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

Keywords: MERS; MERS-CoV; antibody escape mutants; cross-neutralization; multiple strains; receptor-binding domain; spike protein.

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Figures

FIG 1
FIG 1
Construction, characterization, and antigenicity of human and camel MERS-CoV RBD proteins. (A) Schematic diagram of MERS-CoV S1 subunit. Residues 1 to 18, signal peptide. RBD, receptor-binding domain, which contains the identified critical neutralizing domain covering residues 377 to 588. (B) Construction of RBDs of divergent human and camel MERS-CoV strains fused with Fc of human IgG. Residues represent single or multiple mutations in the RBD of representative human MERS-CoV strains in 2012 to 2015, designated 2012-RBD, 2013-RBD, 2014-RBD, and 2015-RBD, or MERS-CoV from camels (Camel-RBD) in comparison with the RBD of prototype strain EMC2012 (EMC-RBD). (C) SDS-PAGE and Western blot analysis of purified rRBD proteins. Nonboiled (nondenatured) or boiled (denatured) samples (5 μg) were subjected to SDS-PAGE (top) or Western blotting (bottom), and the binding was tested using MERS-CoV RBD-specific antibody (1:1,000). The molecular mass markers (in kilodaltons) are indicated on the left. (D) Detection of antigenicity of rRBD proteins by ELISA. ELISA plates were coated with respective human and camel RBD proteins or human Fc (hIgG-Fc) control and then incubated with neutralizing mouse MAb Mersmab1 and human MAbs m336, m337, and m338 (1.25 μg/ml), which recognize conformational epitopes in the RBD of MERS-CoV EMC2012. The data are presented as mean A450 ± standard deviation (SD) (n = 4) of RBDs binding to MAbs.
FIG 2
FIG 2
Detection of binding of human and camel MERS-CoV RBD proteins to DPP4 receptor. (A) Co-IP followed by Western blotting of binding between human and camel RBD proteins and soluble hDPP4 protein or cell-associated hDPP4 in Huh-7 cells. Recombinant RBD proteins were incubated with hDPP4 protein (left) or Huh-7 cell lysates (right) plus protein A beads and then detected for binding using MERS-CoV RBD-specific (1:1,000, top) or DPP4-specific (0.5 μg/ml, bottom) antibodies. The hDPP4 protein only was included as a control. Quantification of binding between rRBD proteins and hDPP4 (B) or cDPP4 (C) protein by ELISA. ELISA plates were coated with hDPP4 or cDPP4 protein (2 μg/ml) and then incubated with dilutions of MERS-CoV RBD proteins or hIgG-Fc control. The data are presented as means ± SD (n = 4) of RBD binding to hDPP4 or cDPP4 protein. (D) Quantification of binding between rRBD proteins and cell-associated hDPP4 receptor by flow cytometry analysis. Huh-7 cells were sequentially incubated with rRBD proteins (40 μg/ml) or hIgG-Fc control and FITC-labeled anti-human IgG antibody, followed by analysis for binding. The data are presented as means ± SD (n = 4) of RBD binding to Huh-7-expressed hDPP4 receptor. MFI, median fluorescence intensity.
FIG 3
FIG 3
Human and camel MERS-CoV RBD proteins induced highly potent cross-reactive antibody responses in immunized mice. PBS was included as a control. Sera from 10 days after the third immunization were tested for IgG (A), IgG1 (B), and IgG2a (C) antibody responses specific to S1 of MERS-CoV prototype strain EMC2012. The antibody titers are expressed as the endpoint dilutions that remain positively detectable, and they are presented as mean antibody titers ± SD for five mice in each group. 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD represent MERS-CoV strains isolated from humans in 2012 to 2015 and from camels, respectively. EMC-RBD, RBD of MERS-CoV prototype strain EMC2012.
FIG 4
FIG 4
Detection of target proteins and infectivity of MERS pseudoviruses. (A) Packaged MERS pseudoviruses were tested for expression of MERS-CoV S and HIV-1 p24 proteins by Western blotting using anti-MERS-CoV RBD (1:1,000, top) and anti-HIV-1 p24 (183-H12-5C, 1:50, bottom) antibodies, respectively. (B) Detection of infectivity of MERS pseudoviruses in DPP4-expressing Huh-7 cells. Vesicular stomatitis virus G glycoprotein (VSV-G) was included as a positive control.
FIG 5
FIG 5
Human and camel MERS-CoV RBD proteins induced highly potent cross-neutralizing antibodies against divergent human and camel MERS pseudoviruses. MERS pseudoviruses expressing S proteins of divergent human and camel MERS-CoV strains isolated from 2012 to 2015 with single or multiple mutations in the RBD were tested for the ability to cross-neutralize MERS-CoV RBD proteins in Huh-7 cells. Sera of mice immunized with EMC-RBD (A), 2012-RBD (B), 2013-RBD (C), 2014-RBD (D), 2015-RBD (E), Camel-RBD (F), or PBS control (A) were collected at 10 days after the third immunization and analyzed. Neutralizing activity was expressed as 50% neutralizing antibody titers (NT50). The data are presented as means ± SD for five mice in each group.
FIG 6
FIG 6
Human and camel MERS-CoV RBD proteins induced highly potent cross-neutralizing antibodies against MAb escape mutants of MERS pseudoviruses. MERS pseudoviruses expressing RBD MAb escape variants were generated as specified above and tested for cross-neutralizing ability of human and camel RBD proteins in Huh-7 cells. Sera of mice immunized with EMC-RBD (A), 2012-RBD (B), 2013-RBD (C), 2014-RBD (D), 2015-RBD (E), Camel-RBD (F), or PBS control (A) were collected at 10 days after the third immunization and analyzed. Neutralizing activity was expressed as NT50, and the data are presented as means ± SD for five mice in each group.
FIG 7
FIG 7
Human and camel MERS-CoV RBD proteins induced cross-neutralizing antibodies against different human MERS-CoVs. Mice were immunized with the indicated RBD or PBS as a control, and sera were collected at 10 days after the third immunization and examined for the presence of antibodies that neutralized MERS-CoV strains EMC2012 and London1-2012 in Vero E6 cells. Neutralizing antibody titers are presented as the reciprocal of the highest dilution of sera that resulted in a complete inhibition of virus infectivity in at least 50% of the wells (NT50). The data are from pooled sera of five mice in each group.
FIG 8
FIG 8
MERS-CoV RBD with multiple mutations of key residues in the RBM exhibited significantly reduced activity of receptor binding and viral entry. (A) Characterization of mutant MERS-CoV RBD proteins. SDS-PAGE (top) and Western blot (bottom) analyses of the purified mutant RBD proteins containing 3 (RBD-FGG) and 5 (RBD-FGGAA) key mutations, respectively, in the RBM. Nonboiled and boiled protein samples (5 μg) were subjected to SDS-PAGE or Western blotting, followed by detection by MERS-CoV RBD-specific antibody (1:1,000). EMC-RBD wild-type (WT) was included as a control. The molecular mass markers (in kilodaltons) are indicated on the left. (B and C) Detection of binding affinity between mutant MERS-CoV RBD proteins and hDPP4 (B) or cDPP4 (C) protein by ELISA. The ELISA plates were coated with hDPP4 or cDPP4 protein (2 μg/ml) and then incubated with different RBDs. The data are presented as means ± SD (n = 4) of RBD binding to hDPP4 or cDPP4 protein. (D) Detection of binding between mutant RBD proteins and Huh-7 cells expressing hDPP4 by flow cytometry analysis. EMC-RBD WT was included as a control. The data are presented as means ± SD (n = 4) of each RBD (40 μg/ml) binding to hDPP4 in Huh-7 cells. MFI, median fluorescence intensity. In panels B to D, three asterisks (***) indicate P values of <0.001 between mutant and WT RBDs. (E) Detection of entry of MERS pseudoviruses expressing S proteins with 3 (L506F, D509G, D510G) or 5 (L506F, D509G, D510G, R511A, E513A) mutations in the RBM. The infectivity of EMC2012 WT pseudovirus in Huh-7 cells was set as 100% entry, and the infectivity of the corresponding mutant pseudovirus was expressed as the percentage of entry (%). ***, P < 0.001 between mutant and WT pseudoviruses.
FIG 9
FIG 9
MERS-CoV RBD with multiple mutations of key residues in the RBM showed reduced antigenicity and neutralizing immunogenicity. (A and B) Detection of the binding between mutant RBD proteins and RBD-specific neutralizing antibodies by ELISA. ELISA plates were precoated with rRBD proteins (1 μg/ml), and binding was detected using RBD-specific neutralizing MAbs Mersmab1 and m336 (1.25 μg/ml) (A), as well as polyclonal antibodies from sera of mice immunized with EMC-RBD wild-type (WT) protein (B). Serum IgG antibody titers are expressed as the endpoint dilutions that remain positively detectable, and the data are presented as means ± SD (n = 4) of each RBD binding to the antibodies. EMC-RBD WT protein was included as a control. **, P < 0.01; ***, P < 0.001 between mutant and WT RBD proteins. (C and D) Detection of neutralizing activity of MERS-CoV RBD-specific neutralizing MAbs Mersmab1 and m336 (C), as well as polyclonal antibodies from sera of mice immunized with EMC-RBD WT protein (D), against the above-described mutant and WT pseudoviruses. ND50 and NT50 represent the 50% neutralizing dose (for MAbs) and 50% neutralizing antibody titers (for sera), respectively. **, P < 0.01; ***, P < 0.001 between mutant and WT pseudoviruses. (E and F) Detection of IgG (E) and neutralizing antibodies (F) induced by MERS-CoV RBD mutant proteins, or EMC-RBD WT protein control, by ELISA and MERS pseudovirus neutralization assay, respectively. Sera from 10 days after the second immunization were tested for IgG antibodies specific to EMC-RBD and neutralizing antibodies against EMC2012 WT pseudovirus. The antibody titers are presented as means ± SD for five mice in each group. The neutralizing antibody titers are expressed as mean NT50 ± SD for five mice in each group. *, P < 0.05; **, P < 0.01 between mutant and WT RBD proteins.
FIG 10
FIG 10
Distribution of RBD mutation residues in the structural model of MERS-CoV S trimer. Based on the structural homology between MERS-CoV RBD (PDB access code 4L3N) and the corresponding domain in the trimeric MHV S (PDB access code 3JCL), the crystal structure of the former was modeled into the cryo-EM structure of the latter. The core structure of MERS-CoV RBD is in cyan, the RBM is in red, and the MERS-CoV RBD residues that have undergone mutations are in blue. The trimeric MHV S protein contains three copies of this domain, with two colored in magenta and the third replaced by MERS-CoV RBD.
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