Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments—The importance of immunofocusing in subunit vaccine design

2.1. Ethics statement

Four- to six-week-old female BALB/c mice and four- to five-month-old female NZW rabbits were used in the study. The animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of the New York Blood Center (Permit Number: 194.15).

2.2. Construction, expression and purification of recombinant proteins

The construction, expression and purification of recombinant MERS-CoV RBD fragments with Fc of human IgG were done as previously described with some modifications [18], [29]. Briefly, genes encoding residues 350-588, 358-588, 367-588, 367-606, and 377-588 of MERS-CoV S protein were respectively amplified by PCR using codon-optimized MERS-CoV S sequences (GenBank: AFS88936.1) as template and inserted into the pFUSE-hIgG1-Fc2 expression vector (hereinafter named Fc; InvivoGen, San Diego, CA). MERS-CoV S1 (residues 18-725) plus a C-terminal His₆ (S1-His) was amplified and inserted into pJW4303 expression vector (Jiangsu Taizhou Haiyuan Protein Biotech, Co., Ltd., China). The recombinant plasmids were transfected into 293T cells (ATCC, Manassas, VA), changed into fresh serum-free DMEM (Invitrogen, Carlsbad, CA) 8 h later, and collected for supernatant containing expressed proteins 72 h post-transfection. The recombinant proteins were purified by Protein A affinity chromatography (GE Healthcare, Piscataway, NJ) for proteins with Fc or Ni-NTA Superflow (Qiagen, Valencia, CA) for proteins with His tag, according to the manufacturers’ instructions.

Human DPP4 ectodomain (residues 39-766) was expressed and purified as previously described [19]. Briefly, recombinant human DPP4 ectodomain containing an N-terminal honeybee melittin signal peptide and a C-terminal His₆ was expressed in insect cells using the Bac-to-Bac expression system (Invitrogen), secreted into cell culture medium, and subsequently purified by Ni-NTA affinity column and Superdex200 gel filtration column (GE Healthcare).

2.3. SDS-PAGE and Western blot

The purified MERS-CoV RBD fragments were analyzed by SDS-PAGE and Western blot as previously described [29], [30]. Briefly, the boiled and nonboiled proteins were separated by 10% Tris–Glycine SDS-PAGE gels, followed by transferring to nitrocellulose membranes. After blocking overnight at 4 °C using 5% non-fat milk in PBST, the blots were incubated for 1 h at room temperature with MERS-CoV S-specific polyclonal antibodies (1:1000). After three washes, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:3000, Invitrogen) for 1 h at room temperature, and the signals were visualized using ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).

2.4. Co-immunoprecipitation assay

The binding of MERS-CoV RBD fragments with DPP4 was performed by co-immunoprecipitation assay as previously described [18]. Briefly, recombinant soluble DPP4 (sDPP4) (10 μg) or DPP4-expressing Huh-7 cell lysates (5 × 10⁷/ml) were respectively incubated with MERS-CoV RBD fragments (10 μg) plus Protein A Sepharose Beads at 4 °C for 1 h, followed by washing with lysis buffer and PBS, and boiling for 10 min. The samples were subjected to SDS-PAGE and Western blot analysis, followed by detection using anti-DPP4 monoclonal antibody (1 μg/ml, R&D Systems, Minneapolis, MN) and MERS-CoV S1-specific polyclonal antibodies (1:1000), respectively.

2.5. Animal vaccination and sample collection

Mice were immunized with MERS-CoV RBD fragments following a previously described protocol [18], [29]. Briefly, mice were prime-vaccinated (subcutaneously, s.c.) with 10 μg/mouse of recombinant MERS-CoV RBD fragments in the presence of Montanide ISA 51 adjuvant (SEPPIC, Fairfield, NJ), and boosted twice with the same immunogen and adjuvant at 3-week (21 days) intervals. Sera from 10 days post-last vaccination (52 days) were heat-inactivated at 56 °C for 30 min, and detected for MERS-CoV S-specific antibody response and neutralizing antibodies. Sera from 0, 10, 31, and 136 days post-initial immunization of S377-588-Fc were also tested for MERS-CoV S-specific antibody response.

The rabbit immunization (Covance Research Products Inc., Denver, PA) followed a protocol similar to that of mice with some modifications. Briefly, rabbits were prime-vaccinated with MERS-CoV S377-588-Fc fragment (250 μg/rabbit) plus Freund's complete adjuvant and then boosted twice at 3-week intervals with the same immunogen (125 μg/rabbit) plus Freund's incomplete adjuvant. Sera were collected and tested as above.

2.6. ELISA

ELISA was carried out to test the binding of MERS-CoV RBD fragments to sDPP4 as previously described [18]. Briefly, 96-well ELISA plates were precoated with sDPP4 (4 μg/ml) overnight at 4 °C and blocked with 2% non-fat milk at 37 °C for 2 h. Diluted MERS-CoV RBD fragments were added to the plates and incubated at 37 °C for 1 h, followed by four washes. Bound antibodies were incubated with HRP-conjugated anti-human IgG (1:3000, Invitrogen) at 37 °C for 1 h. The reaction was visualized by substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Invitrogen) and stopped by 1 N H₂SO₄. The absorbance at 450 nm (A450) was measured by ELISA plate reader (Tecan, San Jose, CA).

The detection of MERS-CoV S-specific antibody responses in collected mouse sera was performed following a protocol similar to that described above, except for replacing the coating antigen with MERS-CoV S1-His protein (1 μg/ml), the primary antibody with serially diluted mouse sera, and the secondary antibody with anti-mouse IgG-HRP (1:3000, Invitrogen) [31].

2.7. Neutralization assay

The standard micro-neutralization assay was used to quantify the neutralizing potential of each of the RBD fragments, as previously described [18], [31], [32]. Briefly, serum samples were diluted at serial 2-fold in 96-well tissue culture plates, and incubated for 1 h at room temperature with ∼100 infectious MERS-CoV/EMC-2012/well before transferring to duplicate wells of Vero E6 cells grown in 96-well tissue culture plates. After 72 h of incubation, when the virus control wells exhibited advanced virus-induced cytopathic effect (CPE), the neutralizing capacity of individual serum samples was assessed by determining the presence or absence of CPE. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited virus-induced CPE in at least 50% of the wells (NT₅₀).

2.8. Statistical analysis

Values are presented as mean with standard deviation (SD). Statistical significance among different vaccination groups was calculated by Student's t-test using Stata statistical software. P values less than 0.05 were considered statistically significant.

3.1. Characterization of MERS-CoV RBD protein fragments

Five human IgG Fc-fused fragments representing the defined RBD residues 358-588, 367-588, 367-606, and 377-588 of MERS-CoV spike protein and the RBD residues 350-588 control were constructed, expressed, and subjected to antigenicity measurement, using the procedures as previously described (Fig. 1B) [18]. All five RBD fragments were expressed at high levels from culture supernatants of transfected 293 T cells and purified to high homogeneity. The C-terminal Fc tag strongly promoted formation of RBD dimers, as evidenced by the almost twice of the molecular weight of non-denatured versus denatured proteins (Fig. 1C, top). These recombinant RBD fragments reacted strongly with polyclonal antibodies specifically raised in mice against recombinant S1 protein of MERS-CoV (Fig. 1C, bottom), suggesting that they are in their native and antigenic conformation.

3.2. Receptor binding affinity of MERS-CoV RBD fragments

Two alternative assays, co-immunoprecipitation and ELISA, were carried out to measure the receptor-binding activities of MERS-CoV RBD fragments. Results from co-immunoprecipitation assay showed that the five MERS-CoV RBD fragments were co-immunoprecipitated with recombinant sDPP4 protein. Two clear bands corresponding to the sizes of DPP4 and respective Fc-fused MERS-CoV RBD fragments were readily revealed with anti-DPP4- and anti-MERS-CoV S1-specific antibodies, whereas a single band with a molecular weight corresponding to DPP4 was detected in the sample containing only sDPP4 (Fig. 2A, left). In addition, each of the five MERS-CoV RBD fragments co-immunoprecipitated cell-associated DPP4 anchored on Huh-7 cells, in which target proteins with anticipated sizes were identified using anti-DPP4- and anti-MERS-CoV S1-specific antibodies, respectively (Fig. 2A, right). Thus, co-immunoprecipitation assay demonstrated that all of the five RBD fragments specifically interact with MERS-CoV's receptor DPP4.

ELISA was then performed by adding MERS-CoV RBD fragments to the plates coated with a fixed amount of sDPP4, followed by identification of the binding affinity based on the OD values at 450 nm. While all of the tested MERS-CoV RBDs were capable of binding to sDPP4, we noted the existence of their differential binding capacity, as shown in Fig. 2B. Among the five RBD fragments, S358-588-Fc, S367-588-Fc and S377-588-Fc possessed significantly higher DPP4-binding affinity than S350-588-Fc and S367-606-Fc at concentrations of 50 and 12.5 μg/ml, respectively. As expected, the control hIgG-Fc protein did not show any specific binding to sDPP4 (Fig. 2B). Therefore, the extended N- or C-terminus of MERS-CoV RBD fragments appears to have negative effect on the receptor-binding activity.

3.3. Immunogenicity of MERS-CoV RBD fragments

To evaluate the immunogenicity of the five MERS-CoV RBD fragments, we immunized mice with each of the RBD fragments according to the dosing strategy described in Materials and Methods, and detected specific responses of total IgG antibody, along with IgG1 and IgG2a subtypes, in serum specimens of immunized mice. ELISA results showed that all five RBD fragments were immunogenic in mice, and induced production of IgG antibodies that specifically bind to MERS-CoV S1 protein without the fusion of Fc. Particularly, sera from mice immunized with S367-588-Fc or S377-588-Fc acquired a stronger binding to MERS-CoV S1 protein than those immunized with the other three RBD fragments, whereas the control samples from PBS-immunized mice revealed negligible binding activity (Fig. 3A), confirming the specificity of such IgG antibody response elicited by these RBD fragments. In addition, the endpoint titration of IgG antibody response revealed that S367-588-Fc and S377-588-Fc were more immunogenic than S350-588-Fc, S358-588-Fc or S367-606-Fc in eliciting MERS-CoV S1-specific IgG antibodies in mice after 3 vaccinations (Fig. 3B). Thus, the extended N- or C-terminus of the MERS-CoV RBD also reduces the immunogenicity of the RBD fragments.

Observation of IgG subtypes revealed that S350-588-Fc, S367-588-Fc and S377-588-Fc were capable of inducing a significantly higher level of IgG1 (Th2) antibody response than S367-606-Fc, while S350-588-Fc and S367-588-Fc elicited IgG1 antibody significantly higher than S358-588-Fc (Fig. 3C). In addition, S350-588-Fc, S358-588-Fc, S367-588-Fc and S377-588-Fc promoted a significantly higher level of IgG2a (Th1) antibody response than S367-606-Fc, whereas the IgG2a antibodies induced by S367-588-Fc were also significantly higher than those induced by S350-588-Fc, S358-588-Fc and S377-588-Fc, respectively (Fig. 3D). The above results suggest that S377-588-Fc is among the RBD fragments that induced high titers of Th1 and Th2 antibody response.

3.4. S377-588-Fc having the best potential to induce highly potent neutralizing antibody responses in vaccinated animals

Neutralizing potentials of the five RBD fragments were initially evaluated in immunized mice. Results from Vero E6-based micro-neutralization showed that each of the five RBD fragments was capable of inducing neutralization antibodies. Among them, S377-588-Fc induced the highest level of neutralizing antibody response on the 10th day after the third immunization (Fig. 4A). As expected, sera from PBS-immunized mice did not show neutralizing activity. Thus, S377-588-Fc has the highest neutralizing potential among the five RBD fragments. Furthermore, S377-588-Fc was used to immunize rabbits to confirm its neutralizing potential in different animal species. Vero E6-based micro-neutralization assay demonstrated that on the 10th day after the third immunization with S377-588-Fc, high titers of neutralizing antibody responses were generated in rabbits (Fig. 4B). Therefore, S377-588-Fc, the shortest among the five RBD fragments, can produce high-titer neutralizing antibody responses in both mice and rabbits, suggesting that the fragment containing residues 377-588 of MERS-CoV spike protein is a critical neutralizing receptor-binding domain.

To elucidate the potential of S377-588-Fc as a vaccine candidate, we immunized mice with this protein for a period of 136 days, and observed the kinetics of antibody responses. As expected, increasing titers of IgG antibodies were found in mouse sera after initial immunization, and reached the highest titers during 31 and 52 days post-immunization. The antibody titers decreased slightly afterwards, but still maintained at relative high levels (Fig. 4C). These data demonstrated that the fragment containing residues 377-588 of MERS-CoV spike protein is able to induce and maintain RBD-specific immune responses in the vaccinated animals for at least four months, confirming its good potential to be further developed as a subunit MERS vaccine.