Immunogenicity of Candidate MERS-CoV DNA Vaccines Based on the Spike Protein

Abstract

MERS-coronavirus is a novel zoonotic pathogen which spread rapidly to >25 countries since 2012. Its apparent endemicity and the wide spread of its reservoir host (dromedary camels) in the Arabian Peninsula highlight the ongoing public health threat of this virus. Therefore, development of effective prophylactic vaccine needs to be urgently explored given that there are no approved prophylactics or therapeutics for humans or animals to date. Different vaccine candidates have been investigated but serious safety concerns remain over protein or full-length spike (S) protein-based vaccines. Here, we investigated the immunogenicity of naked DNA vaccines expressing different fragments of MERS-CoV S protein in mice. We found that plasmids expressing full-length (pS) or S1-subunit (pS1) could induce significant levels of S1-specific antibodies (Abs) but with distinct IgG isotype patterns. Specifically, pS1 immunization elicited a balanced Th1/Th2 response and generally higher levels of all IgG isotypes compared to pS vaccination. Interestingly, only mice immunized with pS1 demonstrated significant S1-specific cellular immune response. Importantly, both constructs induced cross-neutralizing Abs against multiple strains of human and camel origins. These results indicate that vaccines expressing S1-subunit of the MERS-CoV S protein could represent a potential vaccine candidate without the possible safety concerns associated with full-length protein-based vaccines.

Figure 1. MERS-CoV Spike DNA vaccines.

(a) Schematic representation of the generated DNA vaccine constructs. Four constructs were generated including one expressing full length S protein (pS) and three other constructs expressing truncated S protein with deleted cytoplasmic domain (pS∆CD), deleted transmembrane domain (pS∆TM) or deleted S2 subunit (pS1). Numbers indicate amino acids. SP: signal peptide; RBD: receptor-binding domain; TM: transmembrane domain; CD: cytoplasmic domain. (b) In vitro protein expression in cell culture. Vero E6 cells with 80–90% confluency were transfected with the DNA constructs; 48 h later, cell lysates were collected; protein expression was subsequently confirmed by western blot using anti-S1 polyclonal Abs. Arrows indicate band with expected molecular weight. (c) Time-line of immunization regimen.

Figure 2. Humoral immune response induced by MERS-CoV Spike DNA vaccines.

Circulating MERS-CoV S1-specific Abs were determined at 3 weeks post 2^nd boost. End-point titers are shown for (a) total IgG, (b) IgG1, (c) IgG2a and (d) IgG2b isotypes. (e) IgG1/IgG2a ratio was calculated at 3 weeks after 2^nd boosting to determine the type of immune response (Th2 versus Th1) induced by the various constructs. BALB/c mice were i.m. immunized with 100 μg of each construct dissolved in 100 μl PBS on days 0, 14 and 28. A control group was immunized with empty pcDNA vector. Data are shown as mean titer ± s.d. from one experiment out of two independent experiments, with n = 5 mice per treatment group in each experiment. ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05 (one-way ANOVA with Bonferroni post-test).

Figure 3. MERS-CoV Spike-specific memory CD8⁺ T cell responses.

Immunized BALB/c mice were sacrificed at 3 weeks after 2^nd boosting and splenocytes were isolated and re-stimulated ex vivo with synthetic S1 peptides for IFN-γ measurement by ICS. Live CD8⁺ T cells were stained for intracellular IFN-γ. (a) Flow cytometry plots are representatives from one out of two independent experiments. (b) Bar graph represents frequencies of IFN-γ memory CD8⁺ T cells. Data are shown as mean ± s.d from one experiment out of two independent experiments, with n = 3 mice per treatment group in each experiment. **P < 0.01 (one way ANOVA with Bonferroni post-test).

Figure 4. MERS-CoV Spike DNA vaccine induced nAbs.

Neutralization titers were determined as the highest serum dilutions from each individual mouse that completely protected Vero E6 cells in at least 50% of the wells (MNT₅₀). Titers are shown as means from 5 mice per group ± s.d from one experiment out of two independent experiments.