Protective Immunity Against Neospora caninum Infection Induced by 14-3-3 Protein in Mice

Abstract

Neospora caninum is an apicomplexan parasite that infects many mammals and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Our previous studies demonstrated that N. caninum 14-3-3 protein (Nc14-3-3), which is included in N. caninum extracellular vesicles (NEVs), can induce effective immune responses and stimulate cytokine expression in mouse peritoneal macrophages. However, whether Nc14-3-3 has a protective effect and its mechanisms are poorly understood. Here, we evaluated the immune responses and protective effects of Nc14-3-3 against exposure to 2 × 10⁷ Nc-1 tachyzoites. Antibody (IgG, IgGl, and IgG2a) levels and Th1-type (IFN-γ and IL-12) and Th2-type (IL-4 and IL-10) cytokines in mouse serum, survival rates, survival times, and parasite burdens were detected. In the present study, the immunostimulatory effect of Nc14-3-3 was confirmed, as it triggered Th1-type cytokine (IFN-γ and IL-12) production in mouse serum 2 weeks after the final immunization. Moreover, the immunization of C57BL/6 mice with Nc14-3-3 induced high IgG antibody levels and significant increases in CD8⁺ T lymphocytes in the spleens of mice, indicating that the cellular immune response was significantly stimulated. Mouse survival rates and times were significantly prolonged after immunization; the survival rates were 40% for Nc14-3-3 immunization and 60% for NEV immunization, while mice that received GST, PBS, or blank control all died at 13, 9, or 8 days, respectively, after intraperitoneal N. caninum challenge. In addition, qPCR analysis indicated that there was a reduced parasite burden and diminished pathological changes in the mice immunized with Nc14-3-3. Our data demonstrate that vaccination of mice with Nc14-3-3 elicits both cellular and humoral immune responses and provides partial protection against acute neosporosis. Thus, Nc14-3-3 could be an effective antigen candidate for vaccine development for neosporosis.

Keywords: 14-3-3; Neospora caninum; cytokines; extracellular vesicles; immunity.

Figure 1

Measurement of specific IgG antibodies in the sera of immunized mice. Mouse serum was collected from the tail vein plexus before each vaccination, and antibodies were detected by indirect enzyme-linked immunosorbent assays (ELISAs). Determination of specific IgG antibodies in the serum at 0, 2, 4, and 6 weeks. (A) Total IgG; (B) IgG1; and (C) IgG2a. The results are shown as the means of OD450 ± SDs, and significant differences compared with PBS or GST (^**P < 0.01) is indicated by asterisks (^*).

Figure 2

Cytokine production in the serum of mice detected using indirect enzyme-linked immunosorbent assays (ELISAs). Data are expressed as the mean ± SD from three separate experiments. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; and ^****P < 0.0001 for the NEV- and Nc14-3-3-immunized groups vs. the PBS or GST group.

Figure 3

Flow cytometry analysis of T cell subsets. The percentages of CD4⁺ and CD8⁺ T cells in the spleen of immunized mice 2 weeks after the last immunization are shown, and the results are representative of three independent experiments. ^*P < 0.05; ^**P < 0.01 for the NEV- and Nc14-3-3-immunized groups vs. the PBS or GST group.

Figure 4

Survival rates and clinical observations of mice. (A) Survival rates (surviving mice/total mice) of vaccinated mice in response to infection with a dose of 2 × 10⁷ Nc-1 tachyzoites over 40 days. (B). The body weight of mice was recorded daily before death occurred.

Figure 5

Two weeks after the last injection, each mouse was challenged with 2 × 10⁷ Nc-1 tachyzoites. At 5 days post-infection, infected mice were euthanized; the heart, liver, spleen, hung, kidney, and brain were harvested; and parasite loads were measured by qPCR. ^**P < 0.01; and ^***P < 0.001 for the NEV- and Nc14-3-3-immunized groups vs. the PBS or GST group.

Figure 6

Two weeks after the last injection, each mouse was challenged with 2 × 10⁷ Nc-1 tachyzoites. At 5 days post-infection, infected mice were euthanized, and the heart, liver, hung, kidney, and brain were harvested. Pathological changes were observed by H&E staining.