Protective immunity against acute toxoplasmosis in BALB/c mice induced by a DNA vaccine encoding Toxoplasma gondii elongation factor 1-alpha

Abstract

Background: Toxoplasma gondii can infect almost all warm-blood animals including human beings. The high incidence and severe damage that can be caused by T. gondii infection clearly indicates the need for the development of a vaccine. T. gondii elongation factor 1-alpha (TgEF-1α) plays an important role in pathogenesis and host cell invasion for this parasite. The aim of this study was to evaluate the immune protective efficacy of a DNA vaccine encoding TgEF-1α gene against acute T. gondii infection in mice.

Methods: A DNA vaccine (pVAX-EF-1α) encoding T. gondii EF-1a (TgEF-1α) gene was constructed and its immune response and protective efficacy against lethal challenge in BALB/c mice were evaluated.

Results: Mice inoculated with the pVAX-EF-1α vaccine had a high level of specific anti-T. gondii antibodies and produced high levels of IFN-gamma, interleukin (IL)-4, and IL-17. The expression levels of MHC-I and MHC-II molecules as well as the percentages of both CD4(+) and CD8(+) T cells in mice vaccinated with pVAX-EF-1α were significantly increased (p < 0.05), compared with those in all the mice from control groups (blank control, PBS, and pVAXI). Immunization with pVAX-EF-1α significantly (p < 0.05) prolonged mouse survival time to 14.1 ± 1.7 days after challenge infection with the virulent T. gondii RH strain, compared with mice in the control groups which died within 8 days.

Conclusions: DNA vaccination with pVAX-EF-1α triggered strong humoral and cellular responses and induced effective protection in mice against acute T. gondii infection, indicating that TgEF-1α is a promising vaccine candidate against acute toxoplasmosis.

Fig. 1

Identification of the recombinant plasmid with restriction enzyme digestion. Lanes 1, the eukaryotic construct pVAX-EF-1α was double digested by BamH I and Xho I enzymes and the product was resolved by 1 % agarose gel to verify a band of size 1347 bp. (M) Represents DNA Molecular marker

Fig. 2

The phylogenetic tree of amino acid sequences between TgEF-1α and those of EF-1α from other species

Fig. 3

Identification of TgEF-1α in BHK cells by western blot analysis. Lane 1, lysates of BHK cells transfected with pVAX-EF-1α was probed with chicken anti-T. gondii sera. Lane 2, Lysates of BHK cells transfected with empty pVAXI vector probed with chicken anti-T. gondii sera. (M) Pre-stained protein molecular marker

Fig. 4

The dynamics of humoral response in BALB/c mice induced by DNA vaccination. a. Determination of IgG antibodies in the sera of BALB/c mice immunized with pVAX-EF-1α, pVAXI, PBS and Blank controls on weeks 0, 2, 4, 6. Determination of IgG subclass (b) IgG1 and (c) IgG2a, (d) levels of class IgA, (e) levels of class IgM and (f) levels of class IgE in the sera of the immunized BALB/c mice two weeks after the last immunization. Results are expressed as mean of the OD450 ± SD. (n = 5) and statistically significant difference (P < 0.05) and (P < 0.01) are indicated by (*) and (**), respectively

Fig. 5

Cytokine production. Antibody-captured ELISA was used to determine the production levels of (a) IFN-γ, (b) IL-4, (c) IL-17 and (d) TGF-β1, in sera samples (n = 5) collected at weeks 0, 2, 4 and 6, and the comparison results were expressed as means ± SD of pg/ml. The asterisk designates statistically significant differences (* represents p < 0.05; ** represents p < 0.01) between groups. Results presented here were from three independent experiments

Fig. 6

Flow cytometry strategy. Detection of T lymphocyte subpopulation and MHC molecules using flow cytometry technique (CD3 gated), a CD4⁺ T lymphocytes (CD3⁺CD4⁺, region Q2). b CD8⁺ T lymphocytes (CD3⁺CD8⁺, region Q2). c MHC-I molecules (CD3⁺MHC-I, region Q2). d. MHC-II molecules (CD3⁺MHC-II, region Q2)

Fig. 7

Survival curve of mice after challenge infection with Toxoplasma gondii RH strain. Mice were challenged with 10⁴ tachyozoites of the RH strain intraperitoneally two weeks after the third immunization