Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation.

Keywords: CPV; Immune response; Middle East respiratory syndrome coronavirus; Receptor binding domain; Virus-like particles.

Fig. 1

Construction of fused MERS-CoV RBD with VP2 and identification of recombinant RBD-VP2. (A) Schematic diagram for the construction of MERS-CoV RBD and VP2, the MERS-CoV RBD is the spike glycoprotein residues 367–606, and the linker is Gly₄Ser. (B, C, D, E) IFA detection of expression of the chimeric baculoviruses in Sf9 infected cells. Cells were infected with the recombinant baculoviruses in C, E, and were mock infected in B, D. Cells were detected with anti-RBD monoclonal antibody in B and C, and anti-VP2 monoclonal antibody in D and E.

Fig. 2

Characterization and identification of sVLP. (A, B) Western blot analyses of sVLP. Lane 1 lysate from pFastBac1 infected cells served as a control; lane 2 purified RBD recombinant protein expressed in prokaryotic expression system; lane 3 purified VLP of CPV VP2; lane 4 sVLP, analyzed by WB using (A) anti-RBD monoclonal antibody and (B) anti-VP2 monoclonal antibody. (C, D) TEM and IEM analysis of sVLP. (C) sVLP produced by infected Sf9 cells and purified by ammonium sulfate precipitation were stained with 1% sodium phosphotungstate. Bar = 20 nm. (D) The sVLP were incubated with anti-RBD monoclonal antibody and probed using a gold-labeled goat anti-mouse IgG antibody. (E) sVLP hemagglutinates pig erythrocytes.

Fig. 3

Mice immunization procedure, RBD-specific antibody and neutralizing antibodies against MERS-CoV infection. (A) BALB/c (n = 8) were vaccinated IM with 10 μg of sVLP, 10 μg of sVLP and 50 μg of Alum, or 10 μg of sVLP and 50 μg of poly(I:C) adjuvant and the control group was treated with PBS. Blood samples were collected from the orbital vein of mice before immunization and two weeks after each immunization. (B) ELISA results show that immunized mice were able to induce a robust humoral response specific to RBD, with serum IgG titers reaching 1:640 after the second immunization. (C) Immunized mice serum titrations were determined with MERS-pseudotyped virus on Huh 7 cells. The titers were determined as the highest serum dilutions giving a 50% reduction of luciferase activity and are expressed as means+/-SD.

Fig. 4

Flow cytometry for recruitment and/or activation of DCs in lymph nodes from immunized mice. Inguinal lymph nodes were isolated from mice each group 7 days after the second immunization and were stained with mouse anti-CD11c, anti-CD80, and anti-CD86 monoclonal antibodies. Double-positive (A) CD11c⁺CD80⁺, and (B) CD11c⁺CD86⁺ cells were plotted. The data represent the means+/-standard deviation (SD) of double-positive cell percentages. Statistical analysis between the four groups were analyzed by one-way ANOVA (*p < 0.05, **p < 0.01).

Fig. 5

Enzyme-linked immunospot assays of IFN-γ, IL-4 and IL-2 secretion in immunized mice. Splenocytes were isolated from mice and stimulated with purified RBD. Splenocytes secreting (A) IFN-γ or (B) IL-4 and (C) IL-2 were quantitated using ELISpot assay. The data represent the means+/-standard deviation (SD), with units of SFCs per million splenocytes. Statistical analysis between the four groups were analyzed by one-way ANOVA (*p < 0.05, ***p < 0.001, ****p < 0.0001).