Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus

S377-588-Fc at 1 μg was able to induce strong humoral immune responses

To optimize the dose of S377-588-Fc required to induce sufficient antibody responses, we immunized mice with S377-588-Fc at 1, 5, and 20 μg, respectively, and detected specific IgG antibody, as well as IgG1 and IgG2a subtypes, in immunized mouse sera.

As shown in Figure 1A, S377-588-Fc at all 3 test doses was able to induce MERS-CoV S1-specific IgG antibody response, with the antibody rapidly reaching a high level after the 2nd immunization and maintaining similar levels thereafter, suggesting that 2 doses of S377-588-Fc formulated with MF59 adjuvant are sufficient to induce strong antibody responses. As expected, only a background level of IgG antibody response was induced in the adjuvant only group (S377-588-Fc at 0 μg).

We then calculated and compared the endpoint IgG titers from the 3rd sera of mice immunized with the 3 RBD doses. Results, as shown in Figure 1B, revealed that S377-588-Fc at 1 μg induced high levels of IgG antibody response, but such response was significantly increased when the mice received 5 and 20 μg of the S377-588-Fc. Nevertheless, no significant difference was observed for IgG titers in the 5 and 20 μg immunization groups. As expected, mice receiving adjuvant only (S377-588-Fc at 0 μg) failed to induce MERS-CoV S1-specific IgG antibody response (Fig. 1B).

To elucidate the IgG subtypes induced by different doses of S377-588-Fc, we detected IgG1 and IgG2a production using mouse sera from the 3rd immunization. As shown in Figure 2, high titers of IgG1 (Th2) and IgG2a (Th1) antibodies were induced by 1, 5, and 20 μg of S377-588-Fc. However, S377-588-Fc at 5 and 20 μg induced a significantly higher level of IgG1 antibody than that at 1 μg (Fig. 2A). Notably, while no significant difference was revealed between titers of IgG1 and IgG2a antibodies induced by 5 and 20 μg of S377-588-Fc, 5 μg of S377-588-Fc appears to elicit stronger IgG2a antibodies than either 1 or 20 μg (Fig. 2B). As expected, no IgG subtypes were induced in the adjuvant control group (Fig. 2).

The above results suggest that 1 μg of S377-588-Fc is sufficient to induce high titers of RBD-specific antibody responses. Although S377-588-Fc at 5 and 20 µg could induce higher titers of total IgG and IgG1 subtype than those at 1 µg, the increased level of IgG and IgG1 may not necessarily provide stronger neutralizing antibody response that is required for protecting animals from MERS-CoV infection.

S377-588-Fc at 1 μg induced high levels of neutralizing antibody responses, similar to those induced by 5 and 20 μg in immunized mice

To elucidate the neutralizing potential induced by different doses of S377-588-Fc and determine the minimal dose of such an antigen required to elicit strong neutralization against MERS-CoV infection, we investigated neutralizing antibodies in mouse sera from the 3rd immunization based on MERS pseudovirus and live MERS-CoV neutralization assays. Results, as shown in Figure 3, demonstrated that S377-588-Fc at all 3 doses tested was able to induce potent neutralizing antibody titers against infections of MERS pseudovirus in Huh-7 cells (Fig. 3A) and live MERS-CoV in Vero E6 cells (Fig. 3B). There were no significant differences in the neutralizing activity among the sera of mice immunized with S377-588-Fc at 1, 5, and 20 µg, respectively, in the presence of MF59 adjuvant, while the sera from the adjuvant control mice (S377-588-Fc at 0 μg) showed no neutralizing activity (Fig. 3). These data suggest that S377-588-Fc at 1 μg concentration is able to induce sufficient MERS-CoV neutralizing antibodies in the immunized mice.

S377-588-Fc at 1 μg induced high levels of IFN-γ-expressing T cell responses in immunized mice

To compare the cellular immune responses induced by different doses of S377-588-Fc, we immunized mice with the protein at 1, 5, and 20 μg, respectively, and detected MERS-CoV S1-specific IL-2-and IFN-γ-expressing T cells in mouse splenocytes collected from the 3rd immunization. As shown in Figure 4, S377-588-Fc at 1 μg was capable of inducing strong IFN-γ-expressing T cells in both CD4⁺ (Fig. 4A) and CD8⁺ (Fig. 4B) populations, while S377-588-Fc at the increased doses (5 or 20 μg) did not induce higher T cell responses. In addition, S377-588-Fc at 1, 5, or 20 μg failed to elicit strong IL-2-expressing CD4⁺ and CD8⁺ T cells. As expected, the adjuvant control group (S377-588-Fc at 0 μg) induced a background level of specific T cell responses. These results suggest that 1 μg of S377-588-Fc is sufficient to induce potent IFN-γ-expressing T cell responses in immunized mice.

Ethics statement

Four-to six-week-old female BALB/c mice 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).

Recombinant MERS-CoV RBD and S1 proteins

The recombinant S377-588-Fc containing residues 377-588 of MERS-CoV spike plus a C-terminal Fc tag (S377-588-Fc, hereinafter named RBD) was used as the antigen to optimize antigen doses for RBD-based subunit vaccines. The construction, expression and purification of the S377-588-Fc were described previously by fusing the RBD gene with human IgG Fc (InvivoGen, San Diego, CA), expressing the S377-588-Fc protein in 293T cell culture supernatant, and purifying it by Protein A affinity chromatography (GE Healthcare, Piscataway, NJ).^22,23 MERS-CoV S1 protein (residues 18–725) plus a C-terminal His₆ (S1-His) was constructed using the pJW4303 expression vector (Jiangsu Taizhou Haiyuan Protein Biotech, Co., Ltd., China), expressed in 293T cell culture supernatant, and purified using Ni-NTA Superflow (Qiagen, Valencia, CA).^23,24

Animal immunization and sample collection

Mice were immunized with S377-588-Fc as previously described with some modifications.^21,23,35 Briefly, mice were subcutaneously prime-immunized with S377-588-Fc at 1, 5, and 20 μg, respectively, in the presence of MF59 adjuvant,³⁶ and boosted twice with the same immunogen and adjuvant at 3-week intervals. Adjuvant only without antigen (0 μg) was included as the negative control. Sera were collected before immunization and 10 d post-each vaccination to assess MERS-CoV S1-specific antibody responses and neutralizing antibodies. Immunized mice were sacrificed at 10 d after the last immunization, and splenocytes were collected to detect MERS-CoV S1-specific T cell responses.

ELISA

ELISA was used to test MERS-CoV S1-specific antibody responses in mouse sera based on our previously described protocols.^23,37 Briefly, ELISA plates were precoated with MERS-CoV S1-His protein overnight at 4°C, followed by addition of serially diluted sera and incubation at 37°C for 1 h. After four washes with PBST, the plates were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:3000, GE Healthcare), IgG1 (1:2000), or IgG2a (1:5000) (Invitrogen, Carlsbad, CA), respectively, at 37°C for 1 h, and washed 4 times. The reaction was visualized by substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Invitrogen, Carlsbad, CA) and stopped with 1 N H₂SO₄. The absorbance at 450 nm (A450) was measured by ELISA plate reader (Tecan, San Jose, CA).

MERS pseudovirus neutralization assay

This was done as previously described with some modifications.^38,39 Briefly, 293T cells were co-transfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding MERS-CoV (EMC-2012 strain) S protein using the calcium phosphate method. Cells were changed into fresh DMEM 8 h later, and pseudovirus-containing supernatants were harvested 72 h post-transfection for single-cycle infection of Huh-7 cells. The pseudovirus was incubated with serially diluted mouse sera at 37°C for 1 h before adding to the cells preplated in 96-well culture plates. Twenty-four hours later, cells were refed with fresh medium, which was followed by lysing cells 72 h later using cell lysis buffer (Promega, Madison, WI) and transferring the lysates into 96-well luminometer plates. Luciferase substrate (Promega) was added to the plates, and relative luciferase activity was determined in an Infinite 200 PRO Luminator (Tecan). MERS pseudovirus neutralization was calculated and expressed as 50% neutralizing antibody titer, NT₅₀.⁴⁰

Live MERS-CoV neutralization assay

A standard micro-neutralization assay was used to confirm the anti-MERS-CoV neutralizing antibodies as previously described.^23,41 Briefly, mouse sera 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) before transfer to Vero E6 cells. Seventy-two hours later, the neutralizing capacity of serum samples was assessed by determining the presence or absence of virus-induced cytopathic effect (CPE). Neutralizing antibody titers were expressed as the reciprocal of the highest dilution of sera that completely inhibited virus-induced CPE in at least 50% of the wells (NT₅₀).

Intracellular cytokine staining and flow cytometry analysis

MERS-CoV S1-specific cellular immune responses were evaluated in immunized mice by intracellular cytokine staining followed by flow cytometric analysis as previously described.^21,42,43 Briefly, mouse splenocytes (2 × 10⁶) were stimulated with MERS-CoV S1-His protein for 5 h at 37°C with 5% CO₂ in the presence of GolgiPlug™ containing brefeldin A (1 μl/ml; BD Biosciences, San Jose, CA). The cells were stained with conjugated anti-mouse-CD4 (APC) and -CD8 (P-Cy5-5) antibodies for 30 min at 4°C. After washes, the cells were fixed using the Cytofix/Cytoperm™ Kit (BD Biosciences) and stained with anti-mouse-IL-2 (FITC) and -IFN-γ (PE) (BD Biosciences) antibodies for 30 min at 4°C. The stained cells were analyzed using a FACSCalibur (BD Biosciences) and FACSDiva software v.6.1.2 (BD Biosciences).

Statistical analysis

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