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

Abstract

Middle East respiratory syndrome (MERS) is an emerging infectious disease caused by MERS coronavirus (MERS-CoV). The continuous increase of MERS cases has posed a serious threat to public health worldwide, calling for development of safe and effective MERS vaccines. We have previously shown that a recombinant protein containing residues 377-588 of MERS-CoV receptor-binding domain (RBD) fused with human Fc (S377-588-Fc) induced highly potent anti-MERS-CoV neutralizing antibodies in the presence of MF59 adjuvant. Here we optimized the doses of S377-588-Fc using MF59 as an adjuvant in order to elicit strong immune responses with minimal amount of antigen. Our results showed that S377-588-Fc at 1 μg was able to induce in the immunized mice potent humoral and cellular immune responses. Particularly, S377-588-Fc at 1 μg elicited strong neutralizing antibody responses against both pseudotyped and live MERS-CoV similar to those induced at 5 and 20 μg, respectively. These results suggest that this RBD-based subunit MERS vaccine candidate at the dose as low as one μg is sufficiently potent to induce strong humoral and cellular immune responses, including neutralizing antibodies, against MERS-CoV infection, thus providing guidance for determining the optimal dosage of RBD-based MERS vaccines in the future clinical trials and for applying the dose-sparing strategy in other subunit vaccine trials.

Keywords: MERS; MERS-CoV; antigen doses; receptor-binding domain; subunit vaccines.

Figure 1.

Comparison of IgG antibodies induced by S377-588-Fc at different antigen doses. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and sera were assessed for MERS-CoV S1-specific IgG antibody by ELISA. (A) IgG was tested in sera (1:3,200 dilution) of mice before (pre-immune) and 10 d post-each immunization, and the data are presented as mean A450 ± standard deviation (SD) from 5 mice in each group. (B) Sera from 10 d post-last immunization were tested for S1-specific IgG titers and expressed as the endpoint dilutions that remain positively detectable. The data are presented as mean titers ± SD from 5 mice in each group. Significant differences were noted between the groups immunized with 5 or 20 µg, respectively, and 1 µg of MERS-CoV RBD (*).

Figure 2.

Comparison of antibody subtypes induced by S377-588-Fc at different antigen doses. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and sera from 10 d post-last immunization were tested for MERS-CoV S1-specific IgG1 and IgG2a subtypes by ELISA. The titers were expressed as the endpoint dilutions that remained positively detectable, and the values are presented as mean ± SD from 5 mice in each group. Significant differences were noted in the IgG1 responses between the groups immunized with 5 or 20 µg, respectively, and 1 µg of MERS-CoV RBD (*).

Figure 3.

Comparison of neutralization induced by S377-588-Fc at different antigen doses. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and sera from 10 d post-last immunization were tested for neutralization against MERS pseudovirus (A) and live MERS-CoV infection (B) in DPP4-expressing Huh-7 and Vero E6 cells, respectively. MERS pseudovirus neutralization data were expressed as 50% neutralizing antibody titers (NT₅₀), while live MERS-CoV-based neutralizing antibody titers were presented as the reciprocal of the highest dilution of sera that resulted in a complete inhibition of virus-induced CPE in at least 50% of the wells (NT₅₀). The data are presented as mean ± SD from 5 mice in each group.

Figure 4.

Comparison of cellular immune responses induced by S377-588-Fc at different antigen doses. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and splenocytes from 10 d post-last immunization were tested for MERS-CoV S1-specific T cell responses by flow cytometric analysis. The frequencies of IL-2-(upper left corner) and IFN-γ-(bottom right corner) producing cells were expressed as percentages of CD4⁺ (A) or CD8⁺ (B) T cells. The samples were tested in triplicate and presented as mean ± SD from 5 mice in each group.