A Bacterium-like Particle Vaccine Displaying Envelope Proteins of Canine Distemper Virus Can Induce Immune Responses in Mice and Dogs

Abstract

Canine distemper virus (CDV) can cause fatal infections in giant pandas. Vaccination is crucial to prevent CDV infection in giant pandas. In this study, two bacterium-like particle vaccines F₃-GEM and H₄-GEM displaying the trimeric F protein or tetrameric H protein of CDV were constructed based on the Gram-positive enhanced-matrix protein anchor (GEM-PA) surface display system. Electron microscopy and Western blot results revealed that the F or H protein was successfully anchored on the surface of GEM particles. Furthermore, one more bacterium-like particle vaccine F₃ and H₄-GEM was also designed, a mixture consisting of F₃-GEM and H₄-GEM at a ratio of 1:1. To evaluate the effect of the three vaccines, mice were immunized with F₃-GEM, H₄-GEM or F₃ and H₄-GEM. It was found that the level of IgG-specific antibodies and neutralizing antibodies in the F₃ and H₄-GEM group was higher than the other two groups. Additionally, F₃ and H₄-GEM also increased the secretion of Th1-related and Th2-related cytokines. Moreover, F₃ and H₄-GEM induce IgG and neutralizing antibodies' response in dogs. Conclusions: In summary, F₃ and H₄-GEM can provoke better immune responses to CDV in mice and dogs. The bacterium-like particle vaccine F₃ and H₄-GEM might be a potential vaccine candidate for giant pandas against CDV infection.

Keywords: CDV; F protein; H protein; bacterium-like particles; pandas; subunit vaccine.

Figure 1

Construction and identification of F₃-PA and H₄-PA. (A) Schematic of the recombinant baculovirus expressing the F₃-PA and H₄-PA proteins. SP: signal peptide, T4: the trimeric motif; GCN4: the tetrameric motif; linker: (GGGGS)₃; PA: indicates protein anchor. (B) IFA detection of F₃-PA and H₄-PA expression in baculovirus-infected Sf9 cells. Cells were infected with rBV-F₃-PA or rBV-H₄-PA. After 48 h, the cells were detected with a mouse anti-F or H protein polyclonal antibody. (C) Western blot analysis of F or H protein expression in Sf9-infected cells. M: molecular-weight marker, 1: cell sedimentation, 2: supernatant after sonication, and 3: uninfected cells. (D) Western blot identification of F₃ and H₄ proteins with non-reduction treatment; M: molecular-weight marker, 1: supernatant after sonication of F₃-PA; 2: supernatant after sonication of H₄-PA; (E) and analysis of the recombinant baculovirus titers. (E1): F₃-PA; (E2): H₄-PA; (E3,E4): Baculovirus control.

Figure 2

Identification of F3-GEM and H4-GEM. (A) TEM images of MG1363 Lactococcus lactis. (B) TEM images of GEM particles. (C) TEM images of F₃-GEM. (D) TEM images of H₄-GEM. (E) Western blot identification of F or H protein anchored on GEM particles. 1: F₃-GEM; 2: H₄-GEM. (F,G): 0.5 U GEM particles were mixed with 0 mL, 2 mL, 4 mL, 6 mL and 8 mL F or H protein. (H) The amount of F or H protein was determined. 1: GEM; 2: H₄-GEM; 3: F₃-GEMl; and 4–9: different concentrations of BSA proteins.

Figure 3

Serum antibody responses induced by F₃-GEM and H₄-GEM. Serum samples were collected at weeks 0, 2, 4, 6 and 7. Mouse specific total IgG, IgG1 and IgG2a antibody responses were measured using end-point dilution titers (n = 10 mice/group/time point). Data are shown as the mean ± SD and were analyzed by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). (A) Schematic of the experiment. (B,C) Analysis of serum specific total IgG titers induced by different doses of F₃-GEM or H₄-GEM. (D) The IgG titers induced via F₃-GEM, H₄-GEM or F₃ and H₄-GEM. (E): Analysis of IgG1 and IgG2a titers induced by the three vaccines. (F) The ratios of IgG2a/IgG1. (G) The neutralization antibody titers of the three vaccines.

Figure 4

ELISpot analysis of IFN-γ and IL-4 secretion via mouse splenocytes. The splenocytes were collected from each group 7 days after the third immunization was treated and analyzed. (A) The spots on the pictures showed the positive cells that secrete cytokines. The secretions of IFN-γ (B) and IL-4 (C) were measured by using an ELISpot kit. Data are shown as the mean ± SD and were analyzed using one-way ANOVA (**** p < 0.0001).

Figure 5

Detection of cytokines in splenocytes. Splenocytes were harvested from the mice 7 days after the third immunization and restimulated with F or H (10 µg/mL) in culture ex vivo. The concentrations of IFN-γ (A), IL-2 (B), TNF-α (C), IL-4 (D), IL-6 (E) and IL-10 (F) in the supernatant were measured with Meso Scale Discovery based on electrochemiluminescence. Data are shown as the mean ± SD and were analyzed using one-way ANOVA (*** p < 0.001 and **** p < 0.0001).

Figure 6

Antibodies in the immunized dogs. Data are shown as the mean ± SD and were analyzed by one-way ANOVA (* p < 0.05, *** p < 0.001, **** p < 0.0001). (A) The IgG-specific antibody titers in the serum of dogs. (B) The neutralization antibody titers in the serum of dogs.