Protective efficacy of Toxoplasma gondii GRA12 or GRA7 recombinant proteins encapsulated in PLGA nanoparticles against acute Toxoplasma gondii infection in mice

Abstract

Background: Toxoplasma gondii is an apicomplexan parasite that affects the health of humans and livestock, and an effective vaccine is urgently required. Nanoparticles can modulate and improve cellular and humoral immune responses.

Methods: In the current study, poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles were used as a delivery system for the T. gondii dense granule antigens GRA12 and GRA7. BALB/c mice were injected with the vaccines and protective efficacy was evaluated.

Results: Mice immunized with PLGA+GRA12 exhibited significantly higher IgG, and a noticeable predominance of IgG2a over IgG1 was also observed. There was a 1.5-fold higher level of lymphocyte proliferation in PLGA+GRA12-injected mice compared to Alum+GRA12-immunized mice. Higher levels of IFN-g and IL-10 and a lower level of IL-4 were detected, indicating that Th1 and Th2 immune responses were induced but the predominant response was Th1. There were no significant differences between Alum+GRA7-immunized and PLGA+GRA7-immunized groups. Immunization with these four vaccines resulted in significantly reduced parasite loads, but they were lowest in PLGA+GRA12-immunized mice. The survival times of mice immunized with PLGA+GRA12 were also significantly longer than those of mice in the other vaccinated groups.

Conclusion: The current study indicated that T. gondii GRA12 recombinant protein encapsulated in PLGA nanoparticles is a promising vaccine against acute toxoplasmosis, but PLGA is almost useless for enhancing the immune response induced by T. gondii GRA7 recombinant protein.

Keywords: GRA12; GRA7; PLGA; Toxoplasma gondii; nanoparticles; vaccine.

Figure 1

SDS-PAGE and western blotting analysis of T. gondii GRA7 and GRA12 recombinant proteins. (A) SDS-PAGE analysis of recombinant T. gondii GRA7 protein purified via Ni-NTA purification agarose. (B) Western blotting analysis of recombinant T. gondii GRA7-pET-32a protein. (C) SDS-PAGE analysis of recombinant T. gondii GRA12 protein purified via Ni-NTA purification agarose. (D) Western blotting analysis of recombinant T. gondii GRA12 pET-32a protein. The left band represents pET-32a, and the right band represents the recombinant T. gondii GRA12- pET-32a.

Figure 2

Sizes and distributions of recombinant T. gondii GRA7 and GRA12 proteins encapsulated in PLGA NPs as observed via TEM. (A) PLGA+GRA7 NPs (10,000x, scale bar = 1 μm.) (B) PLGA+GRA12 NPs (10,000x, scale bar = 1 μm).

Figure 3

Release characteristics of T. gondii GRA7 and GRA12 proteins using PLGA NPs as a delivery system. Data represent mean ± SD, n = 3 experiments.

Figure 4

Specific antibody responses in mice immunized with Alum+GRA7, PLGA+GRA7, Alum+GRA12, PLGA+GRA12, PBS, Alum, and PLGA. (A) IgG secretion levels. (B) IgG1 secretion levels. (C) IgG2a secretion levels. (D) Levels of IgG1 and IgG2a. Results are represented as 450 nm optical density means ± SD. ^*p< 0.05, ^**p< 0.01, ^***p< 0.001.

Figure 5

Lymphocyte proliferation was evaluated 5 weeks after the last immunization. Five weeks after the last immunization spleens were collected and lymphocyte proliferation responses were assessed. Results represent 450-nm optical density means ± SD. ^*p< 0.05, ^***p< 0.001.

Figure 6

Splenocyte cytokine production after T. gondii lysate TLA stimulation. (A) IFN-γ. (B) IL-4. (C) IL-10. Three mice from each group were analyzed. Data represent means ± SD of three independent experiments. ^*p< 0.05, ^**p< 0.01, ^***p< 0.001.

Figure 7

Parasite loads were analyzed using SYBR-green quantification real-time PCR. Heart, liver, lung, and spleen tissues were collected when symptoms of T. gondii infection became evident, and parasite loads were evaluated. (A) Parasite loads in the heart. (B) Parasite loads in the liver. (C) Parasite loads in the lung. (D) Parasites loads in the spleen. Data are presented as means ± SD of three independent experiments. *p<0.05, **p< 0.01, ***p<0.001.

Figure 8

Survival curves of immunized BALB/c mice after T. gondii challenge. Mice were challenged with 1 × 10³ T. gondii RH strain tachyzoites 5 weeks after the final immunization (n = 10 per group).