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. 2017 Jun;96(6):1441-1447.
doi: 10.4269/ajtmh.16-0548.

Evaluation of the Protective Effect of Deoxyribonucleic Acid Vaccines Encoding Granule Antigen 2 and 5 Against Acute Toxoplasmosis in BALB/c Mice

Xiao Teng Ching  1 , Mun Yik Fong  1 , Yee Ling Lau  1
Affiliations

Evaluation of the Protective Effect of Deoxyribonucleic Acid Vaccines Encoding Granule Antigen 2 and 5 Against Acute Toxoplasmosis in BALB/c Mice

Xiao Teng Ching et al. Am J Trop Med Hyg. 2017 Jun.

Abstract

AbstractToxoplasma gondii infects a broad range of warm-blooded hosts, including humans. Important clinical manifestations include encephalitis in immunocompromised patients as well as miscarriage and fetal damage during early pregnancy. Toxoplasma gondii dense granule antigen 2 and 5 (GRA2 and GRA5) are essential for parasitophorous vacuole development of the parasite. To evaluate the potential of GRA2 and GRA5 as recombinant DNA vaccine candidates, these antigens were cloned into eukaryotic expression vector (pcDNA 3.1C) and evaluated in vaccination experiments. Recombinant DNA vaccines constructed with genes encoding GRAs were validated in Chinese hamster ovary cells before evaluation using lethal challenge of the virulent T. gondii RH strain in BALB/c mice. The DNA vaccines of pcGRA2 and pcGRA5 elicited cellular-mediated immune response with significantly higher levels of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10 (P < 0.05) compared with controls. A mixed T-helper cell 1 (Th1)/Th2 response was associated with slightly prolonged survival. These findings provide evidence that DNA vaccination with GRA2 and GRA5 is associated with Th1-like cell-mediated immune responses. It will be worthwhile to construct recombinant multiantigen combining full-length GRA2 or/and GRA5 with various antigenic proteins such as the surface antigens and rhoptry antigens to improve vaccination efficacy.

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Figures

Figure 1.
Figure 1.
Chinese hamster ovary cells expression of recombinant proteins. Western blot analysis of mammalian cell expressions of pcDNA 3.1C constructs using Xpress mouse monoclonal antibody. Lane 5 contained Prestained Broad Range Protein Marker. Lane 1 contained cell pellet fraction transfected with pcGRA5. Lane 2 contained cell pellet fraction transfected with negative control, pcDNA 3.1C empty vector. Lane 3 contained cell pellet fraction transfected with positive control, pcDNA 3.1/His/lacZ. Lane 4 contained non-transfected cell pellet fraction. Lane 6 contained cell pellet fraction transfected with pcGRA2. The GRA2 and GRA5 protein bands of interest were observed at molecular weights of 30 and 20 kDa (arrow), respectively, compared with the negative control. The positive control expressed β-galactosidase protein with expected size of 120 kDa (arrow).
Figure 2.
Figure 2.
In vitro splenocytes proliferation response in mice. Spleen lymphocytes were harvested from mice immunized with pcGRA2, pcGRA5, pcDNA 3.1C, and phosphate-buffered saline (PBS) 3 weeks after last injection. The splenocytes were cultured and stimulated with total lysate antigen. Proliferative response was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are expressed as mean stimulation index (SI) ± standard deviation (N = 3). Statistical difference is represented by * (P < 0.05) in comparison with the control groups (PBS or pcDNA 3.1C).
Figure 3.
Figure 3.
Interferon-gamma (IFN-γ) and interleukin-2 (IL-2) production by the stimulated splenocytes of the immunized mice. Culture supernatants from the total lysate antigen–stimulated immunized mice splenocytes were collected at 96 and 24 hours postincubation for the evaluation of (A) IFN-γ and (B) IL-2 production, respectively, via enzyme-linked immunosorbent assay. Data are expressed as mean ± standard deviation (N = 3). Statistical difference is represented by * (P < 0.05) in comparison with the control groups (phosphate-buffered saline [PBS] or pcDNA 3.1C).
Figure 4.
Figure 4.
Interleukin-4 (IL-4) and IL-10 production by the stimulated splenocytes of the immunized mice. Culture supernatants from the total lysate antigen–stimulated immunized mice splenocytes were collected at 24 and 72 hours postincubation for the evaluation of (A) IL-4 and (B) IL-10 production, respectively, via enzyme-linked immunosorbent assay. Data are expressed as mean ± standard deviation (N = 3). Statistical difference is represented by * (P < 0.05) in comparison with the control groups (phosphate-buffered saline [PBS] or pcDNA 3.1C).
Figure 5.
Figure 5.
Survival rate of the immunized mice. All four groups of the immunized mice (phosphate-buffered saline [PBS], pcDNA 3.1C, pcGRA2, and pcGRA5) were subjected to lethal challenge with 1,000 live tachyzoites of Toxoplasma gondii-virulent RH strain 3 weeks after the last immunization. Mice immunized with pcGRA2 and pcGRA5 demonstrated a minor increase in their survival days (median survival of 8 days) in comparison to that of the control mice injected with phosphate-buffered saline and pcDNA 3.1C (median survival of 6 and 7 days, respectively). Each group consisted of 10 mice.
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